Water-soluble rhodamine dyes conjugates

ABSTRACT

The present invention provides novel, water-soluble, red-emitting fluorescent rhodamine dyes and red-emitting fluorescent energy-transfer dye pairs, as well as labeled conjugates comprising the same and methods for their use. The dyes, energy-transfer dye pairs and labeled conjugates are useful in a variety of aqueous-based applications, particularly in assays involving staining of cells, protein binding, and/or analysis of nucleic acids, such as hybridization assays and nucleic acid sequencing.

This application is a continuation of application Ser. No. 12/402,634,filed on Mar. 12, 2009, which is a continuation of application Ser. No.11/059,035, filed on Feb. 14, 2005, now U.S. Pat. No. 7,504,496 issuedon Mar. 17, 2009, which is a continuation of application Ser. No.10/007,253, filed on Oct. 24, 2001, which is a division of applicationSer. No. 09/661,206, filed on Sep. 14, 2000, now U.S. Pat. No.6,372,907, issued Apr. 16, 2002, which is a division of application Ser.No. 09/433,093, filed on Nov. 3, 1999, now U.S. Pat. No. 6,191,278,issued Feb. 20, 2001, all of which are incorporated herein by referencein their entireties.

1. FIELD OF THE INVENTION

The present invention relates generally to fluorescent dye compoundsthat are useful as molecular probes. In particular, the presentinvention relates to fluorescent rhodamine dye compounds that arephotostable and highly water-soluble.

2. BACKGROUND OF THE INVENTION

The non-radioactive detection of nucleic acids utilizing fluorescentlabels is an important technology in modern molecular biology. Byeliminating the need for radioactive labels, safety is enhanced and theenvironmental impact and costs associated with reagent disposal isgreatly reduced. Examples of methods utilizing such non-radioactivefluorescent detection include automated DNA sequencing, oligonucleotidehybridization methods, detection of polymerase-chain-reaction products,immunoassays, and the like.

In many applications it is advantageous to employ multiple spectrallydistinguishable fluorescent labels in order to achieve independentdetection of a plurality of spatially overlapping analytes, i.e.,multiplex fluorescent detection. Examples of methods utilizing multiplexfluorescent detection include single-tube multiplex DNA probe assays,PCR, single nucleotide polymorphisms and multi-color automated DNAsequencing. The number of reaction vessels may be reduced therebysimplifying experimental protocols and facilitating the production ofapplication-specific reagent kits. In the case of multi-color automatedDNA sequencing, multiplex fluorescent detection allows for the analysisof multiple nucleotide bases in a single electrophoresis lane therebyincreasing throughput over single-color methods and reducinguncertainties associated with inter-lane electrophoretic mobilityvariations.

Assembling a set of multiple spectrally distinguishable fluorescentlabels useful for multiplex fluorescent detection is problematic.Multiplex fluorescent detection imposes at least six severe constraintson the selection of component fluorescent labels, particularly forapplications requiring a single excitation light source, anelectrophoretic separation, and/or treatment with enzymes, e.g.,automated DNA sequencing. First, it is difficult to find a set ofstructurally similar dyes whose emission spectra are spectrallyresolved, since the typical emission band half-width for organicfluorescent dyes is about 40-80 nanometers (nm). Second, even if dyeswith non-overlapping emission spectra are identified, the set may stillnot be suitable if the respective fluorescent quantum efficiencies aretoo low. Third, when several fluorescent dyes are used concurrently,simultaneous excitation becomes difficult because the absorption bandsof the dyes are usually widely separated. Fourth, the charge, molecularsize, and conformation of the dyes must not adversely affect theelectrophoretic mobilities of the analyte. Fifth, the fluorescent dyesmust be compatible with the chemistry used to create or manipulate theanalyte, e.g., DNA synthesis solvents and reagents, buffers, polymeraseenzymes, ligase enzymes, and the like. Sixth, the dye must havesufficient photostability to withstand laser excitation.

Currently available multiple dye sets suitable for use in four-colorautomated DNA sequencing applications require blue or blue-green laserlight to adequately excite fluorescence emissions from all of the dyesmaking up the set, e.g., argon-ion lasers. As lower cost red lasersbecome available, a need develops for fluorescent dye compounds andtheir nucleic acid conjugates which satisfy the above constraints andare excitable by laser light having a wavelength above about 500 nm.

3. SUMMARY OF THE INVENTION

These and other objects are furnished by the present invention, which inone aspect provides water-soluble, photostable rhodamine dye compoundsthat can be used as labels in a variety of biological and non-biologicalassays. Generally, the rhodamine dye compounds of the invention comprisea rhodamine-type parent xanthene ring substituted at the xanthene C-9carbon with a substituted phenyl ring. The substituted phenyl ringcontains three to five substituents including: an ortho carboxyl orsulfonate group; one or more aminopyridinium (“Pyr⁺”) groups; and onealkylthio, arylthio or heteroarylthio group. The alkylthio, arylthio orheteroarylthio group is believed to be positioned para to the carboxylor sulfonate group, with the remaining positions being substituted withPyr⁺ groups.

The aminopyridinium groups are attached to the phenyl ring at thepyridinium ring nitrogen and may be substituted or unsubstituted at thepyridinium ring carbons with one or more of a wide variety of the sameor different substituents. The substituents may be virtually any group.However, electron-withdrawing groups (e.g., —NO₂, —F, —Cl, —CN, —CF₃,etc.) should not be attached directly to the pyridinium ring carbons, asthese substituents may adversely affect the synthesis of the rhodaminedyes. Electron-withdrawing groups may be included on a substituent aslong as it is spaced away from the pyridinium ring so as to notadversely affect the synthesis of the dyes. Thus, typical pyridiniumring carbon substituents include, but are not limited to —R, —OR, —SR,—NRR, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X,—C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and—C(NR)NRR, where each R is independently hydrogen, (C₁-C₆) alkyl, orheteroalkyl, (C₅-C₁₄) aryl or heteroaryl. The R groups may be furthersubstituted with one or more of the same or different substituents,which are typically selected from the group consisting of —X, —R′, ═O,—OR′, —SR′, ═S, —NR′R′, ═NR′, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO,—NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R′, —C(O)R′, —C(O)X, —C(S)R′,—C(S)X, —C(O)OR′, —C(O)O⁻, —C(S)OR′, —C(O)SR′, —C(S)SR′, —C(O)NR′R′,—C(S)NR′R′ and —C(NR)NR′R′, where each X is independently a halogen(preferably —F or —Cl) and each R′ is independently hydrogen, (C₁-C₆)alkyl or heteroalkyl, (C₅-C₁₄) aryl or heteroaryl. Preferably, thepyridinium ring carbons are unsubstituted. When substituted, the mostpreferred substituents are the same or different (C₁-C₆) alkyls.

The amino group of the aminopyridinium groups is located at the4-position of the pyridinium ring. The amino group may be a primary,secondary or tertiary amino group, but is typically a tertiary amino.The nitrogen substituents are typically (C₁-C₆) alkyl groups orheteroalkyl groups, and may be the same or different. Alternatively, thenitrogen is substituted with an alkyldiyl or heteroalkyldiyl bridgehaving from 2 to 5 backbone atoms such that the substituents and thenitrogen atom taken together form a ring structure, which may besaturated or unsaturated, but is preferably saturated. The bridgesubstituent may be branched or straight-chain, but is preferablystraight-chain, e.g., ethano, propano, butano, etc. The ring structuremay contain, in addition to the nitrogen atom of the aminopyridinium,one or more heteroatoms, which are typically selected from the groupconsisting of O, S and N. When the nitrogen atom is not included in aring structure, the amino group is preferably dimethylamino. When thenitrogen atom is included in a ring structure, the ring is preferably amorpholino or piperazine ring. Particularly preferred Pyr⁺ groups are4-(dimethylamino)pyridinium, 4-(morpholino) pyridinium, and1-methyl-4-piperazinylpyridinium.

The alkylthio, arylthio or heteroarylthio group is attached to thephenyl ring via the sulfur atom and may also be substituted with one ormore of the same or different substituents. The nature of thesubstituents will depend upon whether the group is an alkylthio,arylthio or heteroarylthio. The alkyl chain of an alkylthio group may besubstituted with virtually any substituent, including, but not limitedto, —X, —R, ═O, —OR, —SR, ═S, —NRR, ═NR, —CX₃, —CN, —OCN, —SCN, —NCO,—NCS, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X,—C(S)R, —C(S)X, —C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR,—C(S)NRR and —C(NR)NRR, where each X is independently a halogen(preferably —F or —Cl) and each R is independently hydrogen, (C₁-C₆)alkyl or heteroalkyl, (C₅-C₁₄) aryl or heteroaryl. The R groups may befurther substituted with one or more of the same or differentsubstituents, which are typically selected from the group consisting of—X, —R′, ═O, —OR′, —SR′, ═S, —NR′R′, ═NR′, —CX₃, —CN, —OCN, —SCN, —NCO,—NCS, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R′, —C(O)R′,—C(O)X, —C(S)R′, —C(S)X, —C(O)OR′, —C(O)O⁻, —C(S)OR′, —C(O)SR′,—C(S)SR′, —C(O)NR′R′, —C(S)NR′R′ and —C(NR)NR′R′, where each X isindependently a halogen (preferably —F or —Cl) and each R′ isindependently hydrogen, (C₁-C₆) alkyl or heteroalkyl, (C₅-C₁₄) aryl orheteroaryl.

Due to synthetic constraints, when the group is an arylthio orheteroarylthio, the aryl or heteroaryl rings should not be directlysubstituted with halogens, although halogens may be included in thesubstituent (e.g., a haloalkyl). Thus, when the group is an arylthio ora heteroarylthio, typical substituents include any of the above-listedalkylthio substituents, with the exception of halogen.

The rhodamine dyes of the invention may include a linker L that can beused to conjugate the dyes, preferably by way of covalent attachment, toother compounds or substances, such as peptides, proteins, antibodies,nucleoside/tides, polynucleotides, polymers, particles, etc. Theidentity of linker L will depend upon the nature of the desiredconjugation. For example, the conjugation may be: (i) mediated by ionicinteractions, in which case linker L is a charged group; (ii) mediatedby hydrophobic interactions, in which case L is a hydrophobic moiety,(iii) mediated by covalent attachment, in which case L is a reactivefunctional group (R_(x)) that is either capable of forming a covalentlinkage with another complementary functional group (F_(x)) or iscapable of being activated so as to form a covalent linkage withcomplementary functional group F_(x); or (iv) mediated through the useof pairs of specific binding molecules, such as biotin andavidin/streptavidin, in which case linker L is one member of the pair,e.g., biotin.

Linker L is attached to the rhodamine dyes of the invention at therhodamine-type parent xanthene ring and/or it is included as asubstituent on the alkylthio, arylthio or heteroarylthio groupsubstituting the fully substituted phenyl ring. When linker L isattached to the rhodamine-type parent xanthene ring, it is typicallyattached to a xanthene nitrogen or at the xanthene C4 carbon. Therhodamine dyes may have multiple linking moieties, but preferably haveonly a single linking moiety.

Depending upon the particular application, linker L may be attacheddirectly to the rhodamine dye, or indirectly through one or moreintervening atoms that serve as a spacer. Linker L can be hydrophobic orhydrophilic, long or short, rigid, semirigid or flexible, depending uponthe particular application. When L is positioned at the alkylthio,arylthio or heteroarylthio group, it is preferably attached directly tothe molecule. In this latter embodiment, L is a bond.

The new, fully substituted phenyl rings described herein can be used toreplace the “bottom ring” or “bottom substituent,” i.e., the substituentattached to the xanthene C9 carbon, of virtually any rhodamine dye thatis known in the art or that will be later developed. Thus, the new,fully substituted phenyl rings described herein can be covalentlyattached to the C-9 position of virtually any rhodamine-type parentxanthene ring that is now known or that will be later developed to yielda rhodamine dye without longer absorption and emission maxima and withgreater water-solubility. As the new bottom rings do not deleteriouslyaffect the photostability properties that are characteristic ofrhodamine dyes, the new dyes are also highly photostable. Exemplaryrhodamine-type parent xanthene rings that can comprise the rhodaminedyes of the invention include, by way of example and not limitation, thexanthene rings (“top rings”) of the rhodamine dyes described in U.S.Pat. No. 5,936,087; U.S. Pat. No. 5,750,409; U.S. Pat. No. 5,366,860;U.S. Pat. No. 5,231,191; U.S. Pat. No. 5,840,999; U.S. Pat. No.5,847,162; U.S. application Ser. No. 09/038,191, filed Mar. 10, 1998;U.S. application Ser. No. 09/277,793, filed Mar. 27, 1999; U.S.application Ser. No. 09/325,243, filed Jun. 3, 1999; PCT Publication WO97/36960; PCT Publication WO 99/27020; Sauer et al., 1995, J.Fluorescence 5(3):247-261; Arden-Jacob, 1993, Neue LanwelligeXanthen-Farbstoffe für Fluoreszenzsonden and Farbstoff Laser, VerlagShaker, Germany, and Lee et al., 1992, Nucl. Acids Res.20(10):2471-2483. Preferred rhodamine-type parent xanthene rings arefluorescent.

In another aspect, the invention provides labeled conjugates comprisinga rhodamine dye according to the invention and another molecule orsubstance. The rhodamine dye is conjugated to the other molecule orsubstance, typically via covalent attachment, through linker L, aspreviously described. Once conjugated, the rhodamine dye provides aconvenient fluorescent label for subsequent detection. The rhodaminedyes of the invention can be used to fluorescently label a wide varietyof molecules and substances, including but not limited to, amino acids,peptides, proteins, antibodies, enzymes, receptors, nucleosides/tides,nucleoside/tide analogs, polynucleotides, polynucleotide analogs,nucleic acids, carbohydrates, lipids, steroids, hormones, vitamins,drugs, metabolites, toxins, organic polymers, etc. The dyes can also beused to label particles such as solid phase synthesis substrates,nanoparticles, microspheres or liposomes. In embodiments involvingnanoparticles, microspheres and/or liposomes, the due need not include alinking moiety. It can be incorporated into the various particles duringtheir formation. The molecule or substance may be labeled with one ormore rhodamine dyes of the invention, which may be the same ordifferent.

In one embodiment, a rhodamine dye of the invention is covalentlyconjugated to another dye compound to form an energy-transfer dye pair.The energy-transfer dye pair can be further conjugated to othermolecules or substances, as described above, to provide anenergy-transfer label. The energy-transfer dye pair generally comprisesa donor dye (DD), an acceptor dye (AD), and a linkage linking the donorand acceptor dyes. The donor dye is capable of absorbing light at afirst wavelength and emitting excitation energy in response. Theacceptor dye is capable of absorbing the excitation energy emitted bythe donor dye and fluorescing at a second wavelength in responsethereto. While in many instances the emission wavelength of the donordye and the excitation wavelength of the acceptor dye will overlap, suchoverlap is not required. The acceptor dye need only fluoresce inresponse to the donor dye absorbing light, regardless of the mechanismof action. The linkage serves to facilitate efficient energy transferbetween the donor and acceptor dyes. According to this aspect of theinvention, at least one of the donor and acceptor dyes is a rhodaminedye according to the invention. Preferably, the acceptor dye is arhodamine dye according to the invention and the donor dye is a xanthenedye, most typically a fluorescein dye. The exact nature or identity ofthe donor dye will depend upon the excitation and emission properties ofthe rhodamine acceptor dye, and will be apparent to those having skillin the art. When covalently conjugated to enzymatically-incorporatablenucleotides, such as dideoxynucleotide 5′-triphosphates, i.e.“terminators”, such energy-transfer dyes are ideally suited for use insequencing nucleic acids.

Since the rhodamine dyes of the invention may comprise virtually anyrhodamine-type parent xanthene ring, the dyes cover a broad range of thevisible spectrum, ranging from green to red. Thus, both dyes of anenergy-transfer dye pair may be rhodamine dyes of the invention. In thisembodiment, one dye of the invention acts as the donor and another asthe acceptor, depending upon their spectral properties.

In another embodiment, a rhodamine dye of the invention, or anenergy-transfer dye pair including a rhodamine dye of the invention, iscovalently conjugated to a nucleoside/tide, nucleoside/tide analog,polynucleotide or polynucleotide analog to form a labeled conjugatetherewith. The dye or dye pair is typically conjugated to the nucleobasemoiety of the respective nucleoside/tide, polynucleotide or analog, butmay be conjugated to other portions of the molecule, such as the5′-terminus, 3′-terminus and/or the phosphate ester internucleosidelinkage.

In one preferred embodiment, the labeled conjugate is a labeledpolynucleotide or polynucleotide analog that can be used as a primer forgenerating labeled primer extensions products via template-directedenzymatic synthesis reactions. In another preferred embodiment, thelabeled conjugate is a labeled terminator. When used in conjunction withenzymatically-extendable nucleotides or nucleotide or analogs,appropriate polymerizing enzymes and a primed template nucleic acid,such labeled terminators can be used to generate a series of labeledprimer extension products via template-directed enzymatic synthesis forapplications such as nucleic acid sequencing.

In a final aspect, the invention provides methods of using the rhodaminedyes or energy-transfer dye pairs of the invention to sequence a targetnucleic acid. The method generally comprises forming a series ofdifferently-sized primer extension products labeled with a rhodamine dyeor energy-transfer dye pair of the invention, separating the series ofdifferently-sized labeled extension products, typically based on size,and detecting the separated labeled extension products based on thefluorescence of the label. The sequence of the target nucleic acid isthen assembled according to known techniques.

The series of differently-sized labeled extension products can beconveniently generated by enzymatically extending a primer-target hybridaccording to well-known methods. For example, the series of labeledextension products can be obtained using a primer labeled with arhodamine dye or dye pair of the invention and enzymatically extendingthe labeled primer-target hybrid in the presence of a polymerase, amixture of enzymatically-extendable nucleotides or nucleotide analogscapable of supporting continuous primer extension (e.g., dATP, dGTP,dCTP and dUTP or dTTP) and at least one, typically unlabeled, terminatorthat terminates primer extension upon incorporation (e.g., a ddNTP).Alternatively, the series of labeled extension products can be obtainedusing an unlabeled primer and enzymatically extending the unlabeledprimer-target hybrid in the presence of a polymerase, a mixture ofenzymatically-extendable nucleotides or nucleotide analogs capable ofsupporting continuous primer extension and at least one terminatorlabeled with a rhodamine dye or energy-transfer dye pair of theinvention. In either embodiment, the polymerase serves to extend theprimer with enzymatically-extendable nucleotides or nucleotide analogsuntil a terminator is incorporated, which terminates the extensionreaction. Once terminated, the series of labeled extension products areseparated, typically based on size, and the separated labeled extensionproducts detected based on the fluorescence of the labels.

In a particularly advantageous embodiment of this method, a mixture offour different terminators are used in a single extension reaction. Eachdifferent terminator is capable of terminating primer extension at adifferent template nucleotide, e.g., a mixture of 7-deaza-ddATP, ddCTP,7-deaza-ddGTP and ddTTP or ddUTP, and is labeled with a different,spectrally-resolvable fluorophore, at least one of which is a rhodaminedye or energy-transfer dye pair according to the invention. According tothis embodiment, an unlabeled primer-target nucleic acid hybrid isenzymatically extended in the presence of a polymerase, a mixture ofenzymatically-extendable nucleotides or nucleotide analogs capable ofsupporting continuous primer extension and a mixture of the fourdifferent labeled terminators. Following separation based on size, aseries of separated labeled extension products is obtained in which theemission properties (i.e., color) of each separated extension productreveals the identity of its 3′-terminal nucleotide. In a particularlypreferred embodiment, all of the labeled terminators are excitable usinga single light source.

Alternatively, terminators may be used in the absence ofenzymatically-extendable nucleotides. In this instance, the primer isextended by only a single base. Again, the primer may be labeled, or,alternatively, one or more of the terminators may be labeled.Preferably, a mixture of four different labeled terminators is used, asdescribed above. These “mini sequencing” embodiments are particularlyuseful for identifying polymorphisms in chromosomal DNA or cDNA.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides single color sequencing data obtained with plasmid pGEM,an unlabeled sequencing primer and the labeled terminator6-FAM-196-7-deaza-ddATP on an ABI PRISM Model 310 sequencer (PEBiosystems, Foster City, Calif.).

FIG. 2 provides four-color sequencing data obtained with plasmid pGEM1,an unlabeled sequencing primer and a mixture of four, spectrallyresolvable, 3′-fluoro, labeled terminators: 6-FAM-196-7-deaza-ddATP;5-FAM-dR110-7-deaza-ddGTP; 6-FAM-dION-ddTTP; and 6-FAM-dROX-ddCTP on anABI PRISM Model 310 sequencer (PE Biosystems, Foster City, Calif.).

FIG. 3 provides the fluorescence emission spectra (H₂O) of four dyeswith emission maxima from left to right: 236 (Emax=545 nm), 190(Emax=650 nm), 196 (Emax=661 nm), 232 (Emax=665 nm). The ordinate axisis fluorescence units and the abscissa axis is emission wavelength innm.

FIG. 4 is a plot of a standard curve of the log v. log graph of theaverage fluorescence intensity in detection at FL1 (650-685 nm) vs.pg/ml of the IL-8 peptide by a fluorescence-linked immunosorbent assay.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Abbreviations

The abbreviations used throughout the specification to refer to certainnucleobases, nucleosides and/or nucleotides are those commonly employedin the art and are as indicated below:

Expression Abbreviation adenine A 7-deazaadenine 7-deaza-AN⁶-Δ²-isopentenyladenine 6iA N⁶-Δ²-isopentenyl-2-methylthioadenine2ms6iA cytosine C guanine G 6-thioguanine 6sG 7-deazaguanine 7-deaza-GN²-dimethylguanine 2dmG 7-methylguanine 7mG thymine T 4-thiothymine 4sTuracil U dihydrouracil D 4-thiouracil 4sU base Y Yribonucleoside-5′-triphosphate NTP adenosine-5′-triphosphate ATP7-deazaadenosine-5′-triphosphate 7-deaza-ATP cytidine-5′-triphosphateCTP guanosine-5′-triphosphate GTP 7-deazaguanosine-5′-triphosphate7-deaza-GTP thymidine-5′-triphosphate TTP uridine-5′-triphosphate UTP2′-deoxyribonucleoside-5′-triphosphate dNTP2′-deoxyadenosine-5′-triphosphate dATP2′-deoxy-7-deazaadenosine-5′-triphosphate 7-deaza-dATP2′-deoxycytidine-5′-triphosphate dCTP 2′-deoxyguanosine-5′-triphosphatedGTP 2′-deoxy-7-deazaguanosine-5′-triphosphate 7-deaza-dGTP2′-deoxythymidine-5′-triphosphate dTTP 2′-deoxyuridine-5′-triphosphatedUTP 2′,3′-dideoxyribonucleoside-5′-triphosphate ddNTP2′,3′-dideoxyadenosine-5′-triphosphate ddATP2′,3′-dideoxy-7-deazaadenosine-5′-triphosphate 7-deaza-ddATP2′,3′-dideoxycytidine-5′-triphosphate ddCTP2′,3′-dideoxyguanosine-5′-triphosphate ddGTP2′,3′-dideoxy-7-deazaguanosine-5′-triphosphate 7-deaza-ddGTP2′,3′-dideoxythymidine-5′-triphosphate ddTTP2′,3′-dideoxyuridine-5′-triphosphate ddUTP

5.2 Definitions

In general, the terms used herein to describe the present invention relyon definitions as understood and used by those skilled in the art. Inparticular, chemical structures and substructures are describedaccording to IUPAC recommendations (“Nomenclature of Organic Compounds:A Guide to IUPAC Recommendations 1993, R. Panico, W. H. Powell, andJean-Claude Richer, Eds., Blackwell Science, Ltd., Oxford, U.K.). Asused herein, the following terms are intended to have the followingmeanings:

“Spectrally Resolvable:” means, in reference to a set of fluorescentdyes and/or energy-transfer dye pairs (collectively referred to hereinas “dyes” or “labels”), that the fluorescence emission bands of therespective dyes are sufficiently distinct, i.e., sufficientlynon-overlapping, that the dyes, either alone or when conjugated to othermolecules or substances, are distinguishable from one another on thebasis of their fluorescence signals using standard photodetectionsystems such as photodetectors employing a series of band pass filtersand photomultiplier tubes, charged-coupled devices (CCD), spectrographs,etc., as exemplified by the systems described in U.S. Pat. Nos.4,230,558 and 4,811,218 or in Wheeless at al., 1985, Flow Cytometry:Instrumentation and Data Analysis, pp. 21-76, Academic Press, New York.Preferably, all of the dyes comprising a spectrally resolvable set ofdyes are excitable by a single light source.

“Nucleobase:” refers to a substituted or unsubstitutednitrogen-containing parent heteroaromatic ring of a type that iscommonly found in nucleic acids. Typically, but not necessarily, thenucleobase is capable of forming Watson-Crick and/or Hoogsteen hydrogenbonds with an appropriately complementary nucleobase. Exemplarynucleobases include, but are not limited to, purines such as2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine,N⁶-Δ²-isopentenyladenine (6iA), N⁶-Δ²-isopentenyl-2-methylthioadenine(2ms6iA), N⁶-methyladenine, guanine (G), isoguanine, N²-dimethylguanine(dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG),hypoxanthine and O⁶-methylguanine; 7-deaza-purines such as7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidinessuch as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T),4-thiothymine (4sT), 5,6-dihydrothymine, O⁴-methylthymine, uracil (U),4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indolessuch as nitroindole and 4-methylindole; pyrroles such as nitropyrrole;nebularine; base (Y); etc. Additional exemplary nucleobases can be foundin Fasman, 1989, Practical Handbook of Biochemistry and MolecularBiology, pp. 385-394, CRC Press, Boca Raton, Fla., and the referencescited therein. Preferred nucleobases are purines, 7-deazapurines andpyrimidines. Particularly preferred nucleobases are the normalnucleobases, defined infra, and their common analogs, e.g., 2ms6iA, 6iA,7-deaza-A, D, 2dmG, 7-deaza-G, 7mG, hypoxanthine, 4sT, 4sU and Y.

“Normal Nucleobase:” refers to a nucleobase that is naturally-occurringand encoding, i.e., adenine, cytosine, guanine, thymine or uracil.

“Nucleoside:” refers to a compound consisting of a nucleobase covalentlylinked, typically via a heteroaromatic ring nitrogen, to the C1′ carbonof a pentose sugar. Typical pentose sugars include, but are not limitedto, those pentoses in which one or more of the carbon atoms are eachindependently substituted with one or more of the same or different —R,—OR, —NRR or halogen groups, where each R is independently hydrogen,(C₁-C₆) alkyl or (C₅-C₁₄) aryl. The pentose sugar may be saturated orunsaturated. Exemplary pentose sugars include, but are not limited to,ribose, 2′-deoxyribose, 2′-(C₁-C₆)alkylribose, 2′-(C₁-C₆)alkoxyribose,2′-(C₅-C₁₄)aryloxyribose, 2′,3′-dideoxyribose, 2′,3′-didehydroribose,2′-deoxy-3′-haloribose, 2′-deoxy-3′-fluororibose,2′-deoxy-3′-chlororibose, 2′-deoxy-3′-aminoribose,2′-deoxy-3′-(C₁-C₆)alkylribose, 2′-deoxy-3′-(C₁-C₆)alkoxyribose and2′-deoxy-3′-(C₅-C₁₄)aryloxyribose.

When the nucleobase is a purine or a 7-deazapurine, the pentose sugar isattached to the N9-position of the nucleobase. When the nucleobase is apyrimidine, the pentose sugar is attached to the N1-position of thenucleobase (see, e.g., Komberg and Baker, 1992, DNA Replication, 2^(nd)Ed., Freeman, San Francisco), except for pseudouridine, in which thepentose sugar is attached to the C5 position of the uracil nucleobase.Preferred nucleosides are those in which the nucleobase is a purine, a7-deazapurine, a pyrimidine, a normal nucleobase or a common analogthereof and the pentose sugar is one of the exemplary pentose sugarslisted above.

“Normal Nucleoside:” refers to a compound consisting of a normalnucleobase covalently linked via the N1 (C, T or U) or N9 (A or G)position of the nucleobase to the C1′ carbon of ribose or2′-deoxyribose.

“Nucleoside Analog:” refers to a nucleoside in which the pentose sugaris replaced with a pentose sugar analog. Exemplary pentose sugar analogsinclude, but are not limited to, substituted or unsubstituted furanoseshaving more or fewer than 5 ring atoms, e.g., erythroses and hexoses,and substituted or substituted 3-6 carbon acyclic sugars. One or more ofthe carbon atoms may be independently substituted with one or more ofthe same or different —R, —OR, —NRR or halogen groups, where each R isindependently hydrogen, (C₁-C₆) alkyl or (C₅-C₁₄) aryl.

“Nucleotide:” refers to a nucleoside in which one or more, typicallyone, of the pentose carbons is substituted with a phosphate ester havingthe formula:

where a is an integer from 0 to 4. Preferably, a is 2 and the phosphateester is attached to the 3′- or 5′-carbon of the pentose. Particularlypreferred nucleotides are those which are enzymatically-extendable orenzymatically incorporatable (defined infra).

“Normal Nucleotide:” refers to a normal nucleoside in which the 3′- or5′-carbon of the ribose or 2′-deoxyribose sugar is substituted with aphosphate ester having the formula:

where a is an integer from 0 to 2. Preferred normal nucleotides arethose in which a is 2 and the phosphate ester is attached to the5′-carbon of the ribose (an NTP) or 2′-deoxyribose (a dNTP).

“Nucleotide Analog:” refers to a nucleotide in which the pentose sugarand/or one or more of the phosphate esters is replaced with itsrespective analog. Exemplary pentose sugar analogs are those previouslydescribed in conjunction with nucleoside analogs. Exemplary phosphateester analogs include, but are not limited to, alkylphosphonates,methylphosphonates, phosphoramidates, phosphotriesters,phosphorothioates, phosphorodithioates, phosphoroselenoates,phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates,phosphoroamidates, boronophosphates, etc., including any associatedcounterions, if present.

Also included within the definition of “nucleotide analog” arenucleobase-containing molecules which can be polymerized intopolynucleotide analogs in which the DNA/RNA phosphate ester and/or sugarphosphate ester backbone is replaced with a different type of linkage.Such polynucleotide analogs are described in more detail, infra.

“Enzymatically-Incorporatable Nucleotide or Nucleotide Analog:” refersto a nucleotide or nucleotide analog which is capable of acting as asubstrate for a polymerizing enzyme in a template-directed nucleic acidsynthesis reaction such that it is incorporated by the enzyme into anascent polynucleotide or polynucleotide analog chain. Typicalenzymatically-incorporatable nucleotides and nucleotide analogs arethose in which the sugar is a pentose. Preferredenzymatically-incorporatable nucleotides are those in which thenucleobase is a purine, a 7-deazapurine, a pyrimidine, a normalnucleobase or a common analog thereof and the pentose sugar is apentose-5′-triphosphate, such as NTPs, dNTPs and ddNTPs.

“Enzymatically-Extendable Nucleotide or Nucleotide Analog:” refers to anenzymatically-incorporatable nucleotide or nucleotide analog that, onceincorporated into the nascent polynucleotide or polynucleotide analogchain, supports incorporation of further nucleotides or nucleotideanalogs. Thus, enzymatically-extendable nucleotides or nucleotideanalogs have a hydroxyl group that is capable of forming a covalentlinkage with another, subsequent nucleotide or nucleotide analog.Typical enzymatically-extendable nucleotides and nucleotide analogs arethose in which the sugar is a pentose. Preferredenzymatically-extendable nucleotides are those in which the nucleobaseis a purine, a 7-deazapurine, a pyrimidine, a normal nucleobase or acommon analog thereof and the pentose sugar is a3′-hydroxypentose-5′-triphosphate, such as NTPs and dNTPs.

A mixture of enzymatically-extendable nucleotides or nucleotide analogsis said to support continuous primer extension when the mixture containsan enzymatically-extendable nucleotide or nucleotide analogcomplementary to each base of the template polynucleotide, e.g., amixture of dATP, dGTP, dCTP and dUTP or dTTP.

“Terminator:” refers to an enzymatically-incorporatable nucleotide ornucleotide analog that, once incorporated into the nascentpolynucleotide chain, terminates further chain extension. Typicalterminators are those in which the nucleobase is a purine, a7-deaza-purine, a pyrimidine, a normal nucleobase or a common analogthereof and the sugar moiety is a pentose which includes a3′-substituent that blocks further synthesis, such as a ddNTP.Substituents that block further synthesis include, but are not limitedto, amino, deoxy, halogen, alkoxy and aryloxy groups. Exemplaryterminators include, but are not limited to, those in which thesugar-phosphate ester moiety is 3′-(C₁-C₆)alkylribose-5′-triphosphate,2′-deoxy-3′-(C₁-C₆)alkylribose-5′-triphosphate,2′-deoxy-3′-(C₁-C₆)alkoxyribose-5′-triphosphate,2′-deoxy-3′-(C₅-C₁₄)aryloxyribose-5′-triphosphate,2′-deoxy-3′-haloribose-5′-triphosphate,2′-deoxy-3′-aminoribose-5′-triphosphate,2′,3′-dideoxyribose-5′-triphosphate or2′,3′-didehydroribose-5′-triphosphate.

“Nucleoside/tide:” refers to a nucleoside and/or a nucleotide and/or amixture thereof.

“Polynucleotide:” refers to a linear polymeric chain of nucleosidemonomer units that are covalently connected to one another by phosphateester internucleoside linkages. Unless stated otherwise,“polynucleotide” as used herein includes polymers of any length,including oligonucleotides, polynucleotides and nucleic acids as thoseterms are commonly used in the art. Where polynucleotides of specificsize ranges are intended, the number of monomer units is specificallydelineated. Thus, polynucleotides according to the invention can rangein size from a few monomer units (e.g., 4 to 40), to several hundreds ofmonomer units, to several thousands of monomer units, or even moremonomer units. Whenever a polynucleotide is represented by a sequence ofletters, e.g. “ATGCCTG,” it will be understood that the sequence ispresented in the 5′→3′ direction. 2′-Deoxyribopolynucleotides arepreceded with the letter “d,” e.g. “d(ATGCCTG).”

Polynucleotides may be comprised of a single type of sugar moiety, as inthe case of RNA and DNA, or mixtures of different sugar moieties, as inthe case of RNA/DNA chimeras. Preferred polynucleotides areribopolynucleotides and 2′-deoxyribopolynucleotides according to thestructural formulae below:

wherein:

-   -   each B is independently a nucleobase, preferably a purine, a        7-deazapurine, a pyrimidine, a normal nucleobase or a common        analog thereof;    -   each m defines the length of the respective polynucleotide and        can range from zero to thousands, to tens of thousands, or even        more;    -   each R is independently selected from the group consisting of        hydrogen, halogen, —R″, —OR″, and —NR″R″, where each R″ is        independently (C₁-C₆) alkyl or (C₅-C₁₄) aryl, or two adjacent Rs        are taken together to form a bond such that the ribose sugar is        2′,3′-didehydroribose; and    -   each R′ is independently hydroxyl or

where a is zero, one or two.

In the preferred ribopolynucleotides and 2′-deoxyribopolynucleotidesillustrated above, the nucleobases B are covalently attached to the C1′carbon of the sugar moiety as previously described.

“Polynucleotide Analog:” refers to a polynucleotide in which at leastone nucleoside monomer unit is a nucleoside analog and/or at least onephosphate ester internucleoside linkage is a phosphate ester analog, aspreviously defined. Also included within the definition ofpolynucleotide analogs are polymers in which the phosphate ester and/orsugar phosphate ester internucleoside linkages are replaced with othertypes of linkages, such as N-(2-aminoethyl)-glycine amides and otheramides (see, e.g., Nielsen et al., 1991, Science 254:1497-1500; WO92/20702; U.S. Pat. No. 5,719,262; U.S. Pat. No. 5,698,685; morpholinos(see U.S. Pat. No. 5,698,685; U.S. Pat. No. 5,378,841; U.S. Pat. No.5,185,144); carbamates (see Stirchak & Summerton, 1987, J. Org. Chem.52:4202); methylene(methylimino) (see Vasseur et al., 1992, J. Am. Chem.Soc. 114:4006); 3′-thioformacetals (see Jones et al., 1993, J. Org.Chem. 58:2983); sulfamates (see U.S. Pat. No. 5,470,967); and others(see, e.g., U.S. Pat. No. 5,817,781; Frier & Altman, 1997, Nucl. AcidsRes. 25:4429 and the references cited therein).

“Alkyl:” refers to a saturated or unsaturated, branched, straight-chainor cyclic monovalent hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane, alkene oralkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkenyl”and/or “alkynyl” is used, as defined below. In preferred embodiments,the alkyl groups are (C₁-C₆) alkyl.

“Alkanyl:” refers to a saturated branched, straight-chain or cyclicalkyl radical derived by the removal of one hydrogen atom from a singlecarbon atom of a parent alkane. Typical alkanyl groups include, but arenot limited to, methanyl; ethenyl; propanyls such as propan-1-yl,propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such asbutan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like. Inpreferred embodiments, the alkanyl groups are (C₁-C₆) alkanyl.

“Alkenyl:” refers to an unsaturated branched, straight-chain or cyclicalkyl radical having at least one carbon-carbon double bond derived bythe removal of one hydrogen atom from a single carbon atom of a parentalkene. The radical may be in either the cis or trans conformation aboutthe double bond(s). Typical alkenyl groups include, but are not limitedto, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, prop-2-en-2-yl, cycloprop-1-en-1-yl;cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl,2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl,cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like. Inpreferred embodiments, the alkenyl group is (C₂-C₆) alkenyl.

“Alkynyl” refers to an unsaturated branched, straight-chain or cyclicalkyl radical having at least one carbon-carbon triple bond derived bythe removal of one hydrogen atom from a single carbon atom of a parentalkyne. Typical alkynyl groups include, but are not limited to, ethynyl;propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such asbut-1-yn-1-yl, but-3-yn-1-yl, etc.; and the like. In preferredembodiments, the alkynyl group is (C₂-C₆) alkynyl.

“Alkyldiyl:” refers to a saturated or unsaturated, branched,straight-chain or cyclic divalent hydrocarbon radical derived by theremoval of one hydrogen atom from each of two different carbon atoms ofa parent alkane, alkene or alkyne, or by the removal of two hydrogenatoms from a single carbon atom of a parent alkane, alkene or alkyne.The two monovalent radical centers or each valency of the divalentradical center can form bonds with the same or different atoms. Typicalalkyldiyls include, but are not limited to methandiyl; ethyldiyls suchas ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl;propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl,propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl,prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,prop-1-en-1,3-diyl cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,buta-1,3-dien-1,3-diyl, cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl,cyclobut-2-en-1,2-diyl, cyclobuta-1,3-dien-1,2-diyl,cyclobuta-1,3-dien-1,3-diyl, but-1-yn-1,3-diyl, but-1-yn-1,4-diyl,buta-1,3-diyn-1,4-diyl, etc.; and the like. Where specific levels ofsaturation are intended, the nomenclature alkanyldiyl, alkenyldiyland/or alkynyldiyl is used. In preferred embodiments, the alkyldiylgroup is (C₁-C₆) alkyldiyl. Also preferred are saturated acyclicalkanyldiyl radicals in which the radical centers are at the terminalcarbons, e.g., methandiyl (methano); ethan-1,2-diyl(ethano);propan-1,3-diyl(propano); butan-1,4-diyl(butano); and the like (alsoreferred to as alkylenos, defined infra).

“Alkyleno:” refers to a straight-chain alkyldiyl radical having twoterminal monovalent radical centers derived by the removal of onehydrogen atom from each of the two terminal carbon atoms ofstraight-chain parent alkane, alkene or alkyne. Typical alkyleno groupsinclude, but are not limited to, methano; ethylenos such as ethano,etheno, ethyno; propylenos such as propano, prop[1]eno, propa[1,2]dieno,prop[1]yno, etc.; butylenos such as butano, but[1]eno, but[2]eno,buta[1,3]dieno, but[1]yno, but[2]yno, but[1,3]diyno, etc.; and the like.Where specific levels of saturation are intended, the nomenclaturealkano, alkeno and/or alkyno is used. In preferred embodiments, thealkyleno group is (C₁-C₆) alkyleno.

“Heteroalkyl, Heteroalkanyl, Heteroalkenyl, Heteroalkanyl,Heteroalkyldiyl and Heteroalkyleno:” refer to alkyl, alkanyl, alkenyl,alkynyl, alkyldiyl and alkyleno radicals, respectively, in which one ormore of the carbon atoms are each independently replaced with the sameor different heteroatomic groups. Typical heteroatomic groups which canbe included in these radicals include, but are not limited to, —O—, —S—,—O—O—, —S—S—, —O—S—, —NR′—, ═N—N═, —N═N—, —N(O)N—, —N═N—NR′—, —PH—,—P(O)₂—, —O—P(O)₂—, —SH₂—, —S(O)₂—, —SnH₂— an the like, where each R′ isindependently hydrogen, alkyl, alkanyl, alkenyl, alkynyl, aryl,arylaryl, arylalkyl, heteroaryl, heteroarylalkyl orheteroaryl-heteroaryl as defined herein.

“Acyclic Heteroatomic Bridge:” refers to a divalent bridge in which thebackbone atoms are exclusively heteroatoms. Typical acyclic heteroatomicbridges include, but are not limited to, any of the various heteroatomicgroups listed above, either alone or in combinations.

“Parent Aromatic Ring System:” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, indane, indene, phenalene, etc. Typical parent aromaticring systems include, but are not limited to, aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexylene, as-indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octane, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene,and the like.

“Aryl:” refers to a monovalent aromatic hydrocarbon radical derived bythe removal of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, radicals derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. In preferredembodiments, the aryl group is (C₅-C₁₄) aryl, with (C₅-C₁₀) being evenmore preferred. Particularly preferred aryls are phenyl and naphthyl.

“Aryldiyl:” refers to a divalent aromatic hydrocarbon radical derived bythe removal of one hydrogen atom from each of two different carbon atomsof a parent aromatic ring system or by the removal of two hydrogen atomsfrom a single carbon atom of a parent aromatic ring system. The twomonovalent radical centers or each valency of the divalent center canform bonds with the same or different atom(s). Typical aryldiyl groupsinclude, but are not limited to, divalent radicals derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorine, hexacene,hexaphene, hexylene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene, and the like. In preferred embodiments, the aryldiylgroup is (C₅-C₁₄) aryldiyl, with (C₅-C₁₀) being even more preferred. Themost preferred aryldiyl groups are divalent radicals derived frombenzene and naphthalene, especially phena-1,4-diyl, naphtha-2,6-diyl andnaphtha-2,7-diyl.

“Aryleno:” refers to a divalent bridge radical having two adjacentmonovalent radical centers derived by the removal of one hydrogen atomfrom each of two adjacent carbon atoms of a parent aromatic ring system.Attaching an aryleno bridge radical, e.g. benzeno, to a parent aromaticring system, e.g. benzene, results in a fused aromatic ring system, e.g.naphthalene. The bridge is assumed to have the maximum number ofnon-cumulative double bonds consistent with its attachment to theresultant fused ring system. In order to avoid double-counting carbonatoms, when an aryleno substituent is formed by taking together twoadjacent substituents on a structure that includes alternativesubstituents, the carbon atoms of the aryleno bridge replace thebridging carbon atoms of the structure. As an example, consider thefollowing structure:

wherein:

-   -   R¹, when taken alone is hydrogen, or when taken together with R²        is (C₅-C₁₄) aryleno; and

R², when taken alone is hydrogen, or when taken together with R¹ is(C₅-C₁₄) aryleno.

When R¹ and R² are each hydrogen, the resultant compound is benzene.When R¹ taken together with R² is C₆ aryleno (benzeno), the resultantcompound is naphthalene. When R¹ taken together with R² is C₁₀ aryleno(naphthaleno), the resultant compound is anthracene or phenanthrene.Typical aryleno groups include, but are not limited to, aceanthryleno,acenaphthyleno, acephenanthtyleno, anthraceno, azuleno, benzeno (benzo),chryseno, coroneno, fluorantheno, fluoreno, hexaceno, hexapheno,hexyleno, as-indaceno, s-indaceno, indeno, naphthalene (naphtho),octaceno, octapheno, octaleno, ovaleno, penta-2,4-dieno, pentaceno,pentaleno, pentapheno, peryleno, phenaleno, phenanthreno, piceno,pleiadeno, pyreno, pyranthreno, rubiceno, triphenyleno, trinaphthaleno,and the like. Where a specific connectivity is intended, the involvedbridging carbon atoms (of the aryleno bridge) are denoted in brackets,e.g., [1,2]benzeno ([1,2]benzo), [1,2]naphthaleno, [2,3]naphthaleno,etc. Thus, in the above example, when R¹ taken together with R² is[2,3]naphthaleno, the resultant compound is anthracene. When R¹ takentogether with R² is [1,2]naphthaleno, the resultant compound isphenanthrene. In a preferred embodiment, the aryleno group is (C₅-C₁₄),with (C₅-C₁₀) being even more preferred.

“Arylaryl:” refers to a monovalent hydrocarbon radical derived by theremoval of one hydrogen atom from a single carbon atom of a ring systemin which two or more identical or non-identical parent aromatic ringsystems are joined directly together by a single bond, where the numberof such direct ring junctions is one less than the number of parentaromatic ring systems involved. Typical arylaryl groups include, but arenot limited to, biphenyl, triphenyl, phenyl-naphthyl, binaphthyl,biphenyl-naphthyl, and the like. When the number of carbon atomscomprising an arylaryl group is specified, the numbers refer to thecarbon atoms comprising each parent aromatic ring. For example, (C₅-C₁₄)arylaryl is an arylaryl group in which each aromatic ring comprises from5 to 14 carbons, e.g., biphenyl, triphenyl, binaphthyl, phenylnaphthyl,etc. Preferably, each parent aromatic ring system of an arylaryl groupis independently a (C₅-C₁₄) aromatic, more preferably a (C₅-C₁₀)aromatic. Also preferred are arylaryl groups in which all of the parentaromatic ring systems are identical, e.g., biphenyl, triphenyl,binaphthyl, trinaphthyl, etc.

“Biaryl:” refers to an arylaryl radical having two identical parentaromatic systems joined directly together by a single bond. Typicalbiaryl groups include, but are not limited to, biphenyl, binaphthyl,bianthracyl, and the like. Preferably, the aromatic ring systems are(C₅-C₁₄) aromatic rings, more preferably (C₅-C₁₀) aromatic rings. Aparticularly preferred biaryl group is biphenyl.

“Arylalkyl:” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or spacarbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. Where specific alkyl moieties are intended, the nomenclaturearylalkanyl, arylakenyl and/or arylalkynyl is used. In preferredembodiments, the arylalkyl group is (C₆-C₂₀) arylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₆) andthe aryl moiety is (C₅-C₁₄). In particularly preferred embodiments thearylalkyl group is (C₆-C₁₃), e.g., the alkanyl, alkenyl or alkynylmoiety of the arylalkyl group is (C₁-C₃) and the aryl moiety is(C₅-C₁₀).

“Parent Heteroaromatic Ring System:” refers to a parent aromatic ringsystem in which one or more carbon atoms (and any necessary associatedhydrogen atoms) are each independently replaced with the same ordifferent heteroatom. Typical heteroatoms to replace the carbon atomsinclude, but are not limited to, N, P, O, S, Si, etc. Specificallyincluded within the definition of “parent heteroaromatic ring systems”are fused ring systems in which one or more rings are aromatic and oneor more of the rings are saturated or unsaturated, such as, for example,arsindole, chromane, chromene, indole, indoline, xanthene, etc. Typicalparent heteroaromatic ring systems include, but are not limited to,arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan,imidazole, indazole, indole, indoine, indolizine, isobenzofuran,isochromene, isoindole, isoindoline, isoquinoline, isothiazole,isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike.

“Heteroaryl:” refers to a monovalent heteroaromatic radical derived bythe removal of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, radicals derived from acridine, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. In preferred embodiments,the heteroaryl group is a 5-14 membered heteroaryl, with 5-10 memberedheteroaryl being particularly preferred. The most preferred heteroarylradicals are those derived from parent heteroaromatic ring systems inwhich any ring heteroatoms are nitrogens, such as imidazole, indole,indazole, isoindole, naphthyridine, pteridine, isoquinoline,phthalazine, purine, pyrazole, pyrazine, pyridazine, pyridine, pyrrole,quinazoline, quinoline, etc.

“Heteroaryldiyl:” refers to a divalent heteroaromatic radical derived bythe removal of one hydrogen atom from each of two different atoms of aparent heteroaromatic ring system or by the removal of two hydrogenatoms from a single atom of a parent heteroaromatic ring system. The twomonovalent radical centers or each valency of the single divalent centercan form bonds with the same or different atom(s). Typicalheteroaryldiyl groups include, but are not limited to, divalent radicalsderived from acridine, arsindole, carbazole, β-carboline, chromane,chromene, cinnoline, furan, imidazole, indazole, indole, indoline,indolizine, isobenzofuran, isochromene, isoindole, isoindoline,isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole,oxazole, perimidine, phenanthridine, phenanthroline, phenazine,phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine,pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline,quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene,triazole, xanthene, and the like. In preferred embodiments, theheteroaryldiyl group is 5-14 membered heteroaryldiyl, with 5-10 memberedbeing particularly preferred. The most preferred heteroaryldiyl groupsare divalent radicals derived from parent heteroaromatic ring systems inwhich any ring heteroatoms are nitrogens, such as imidazole, indole,indazole, isoindole, naphthyridine, pteridine, isoquinoline,phthalazine, purine, pyrazole, pyrazine, pyridazine, pyridine, pyrrole,quinazoline, quinoline, etc.

“Heteroaryleno:” refers to a divalent bridge radical having two adjacentmonovalent radical centers derived by the removal of one hydrogen atomfrom each of two adjacent atoms of a parent heteroaromatic ring system.Attaching a heteroaryleno bridge radical, e.g. pyridino, to a parentaromatic ring system, e.g. benzene, results in a fused heteroaromaticring system, e.g., quinoline. The bridge is assumed to have the maximumnumber of non-cumulative double bonds consistent with its attachment tothe resultant fused ring system. In order to avoid double-counting ringatoms, when a heteroaryleno substituent is formed by taking together twoadjacent substituents on a structure that includes alternativesubstituents, the ring atoms of the heteroaryleno bridge replace thebridging ring atoms of the structure. As an example, consider thefollowing structure:

wherein:

-   -   R¹, when taken alone is hydrogen, or when taken together with R²        is 5-14 membered heteroaryleno; and

R², when taken alone is hydrogen, or when taken together with R¹ is 5-14membered heteroaryleno;

When R¹ and R² are each hydrogen, the resultant compound is benzene.When R¹ taken together with R² is a 6-membered heteroaryleno pyridino),the resultant compound is isoquinoline, quinoline or quinolizine. WhenR¹ taken together with R² is a 10-membered heteroaryleno (e.g.,isoquinoline), the resultant compound is, e.g., acridine orphenanthridine. Typical heteroaryleno groups include, but are notlimited to, acridino, carbazolo, β-carbolino, chromeno, cinnolino,furan, imidazolo, indazoleno, indoleno, indolizino, isobenzofurano,isochromeno, isoindoleno, isoquinolino, isothiazoleno, isoxazoleno,naphthyridino, oxadiazoleno, oxazoleno, perimidino, phenanthridino,phenanthrolino, phenazino, phthalazino, pteridino, purino, pyrano,pyrazino, pyrazoleno, pyridazino, pyridino, pyrimidino, pyrroleno,pyrrolizino, quinazolino, quinolino, quinolizino, quinoxalino,tetrazoleno, thiadiazoleno, thiazoleno, thiopheno, triazoleno, xantheno,and the like. Where a specific connectivity is intended, the involvedbridging atoms (of the heteroaryleno bridge) are denoted in brackets,e.g., [1,2]pyridino, [2,3]pyridino, [3,4]pyridino, etc. Thus, in theabove example, when R¹ taken together with R² is [1,2]pyridino, theresultant compound is quinolizine. When R¹ taken together with R² is[2,3]pyridino, the resultant compound is quinoline. When R¹ takentogether with R² is [3,4]pyridino, the resultant compound isisoquinoline. In preferred embodiments, the heteroaryleno group is 5-14membered heteroaryleno, with 5-10 membered being even more preferred.The most preferred heteroaryleno radicals are those derived from parentheteroaromatic ring systems in which any ring heteroatoms are nitrogens,such as imidazolo, indolo, indazolo, isoindolo, naphthyridino,pteridino, isoquinolino, phthalazino, purino, pyrazolo, pyrazino,pyridazino, pyridino, pyrrolo, quinazolino, quinolino, etc.

“Heteroaryl-Heteroaryl:” refers to a monovalent heteroaromatic radicalderived by the removal of one hydrogen atom from a single atom of a ringsystem in which two or more identical or non-identical parentheteroaromatic ring systems are joined directly together by a singlebond, where the number of such direct ring junctions is one less thanthe number of parent heteroaromatic ring systems involved. Typicalheteroaryl-heteroaryl groups include, but are not limited to, bipyridyl,tripyridyl, pyridylpurinyl, bipurinyl, etc. When the number of ringatoms are specified, the numbers refer to the number of atoms comprisingeach parent heteroaromatic ring systems. For example, 5-14 memberedheteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which eachparent heteroaromatic ring system comprises from 5 to 14 atoms, e.g.,bipyridyl, tripyridyl, etc. Preferably, each parent heteroaromatic ringsystem is independently a 5-14 membered heteroaromatic, more preferablya 5-10 membered heteroaromatic. Also preferred are heteroaryl-heteroarylgroups in which all of the parent heteroaromatic ring systems areidentical. The most preferred heteroaryl-heteroaryl radicals are thosein which each heteroaryl group is derived from parent heteroaromaticring systems in which any ring heteroatoms are nitrogens, such asimidazole, indole, indazole, isoindole, naphthyridine, pteridine,isoquinoline, phthalazine, purine, pyrazole, pyrazine, pyridazine,pyridine, pyrrole, quinazoline, quinoline, etc.

“Biheteroaryl:” refers to a heteroaryl-heteroaryl radical having twoidentical parent heteroaromatic ring systems joined directly together bya single bond. Typical biheteroaryl groups include, but are not limitedto, bipyridyl, bipurinyl, biquinolinyl, and the like. Preferably, theheteroaromatic ring systems are 5-14 membered heteroaromatic rings, morepreferably 5-10 membered heteroaromatic rings. The most preferredbiheteroaryl radicals are those in which the heteroaryl groups arederived from a parent heteroaromatic ring system in which any ringheteroatoms are nitrogens, such as biimidazolyl, biindolyl, biindazolyl,biisoindolyl, binaphthyridinyl, bipteridinyl, biisoquinolinyl,biphthalazinyl, bipurinyl, bipyrazolyl, bipyrazinyl, bipyridazinyl,bipyridinyl, bipyrrolyl, biquinazolinyl, biquinolinyl, etc.

“Heteroarylalkyl:” refers to an acyclic alkyl radical in which one ofthe hydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Where specific alkylmoieties are intended, the nomenclature heteroarylalkanyl,heteroarylakenyl and/or heterorylalkynyl is used. In preferredembodiments, the heteroarylalkyl group is a 6-20 memberedheteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of theheteroarylalkyl is 1-6 membered and the heteroaryl moiety is a5-14-membered heteroaryl. In particularly preferred embodiments, theheteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., the alkanyl,alkenyl or alkynyl moiety is 1-3 membered and the heteroaryl moiety is a5-10 membered heteroaryl.

“Substituted:” refers to a radical in which one or more hydrogen atomsare each independently replaced with the same or differentsubstituent(s). Typical substituents include, but are not limited to,—X, —R, —O⁻, ═O, —OR, —O—OR, —SR, —S⁻, ═S, —NRR, ═NR, perhalo (C₁-C₆)alkyl, —CX₃, —CF₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂, ═N₂,—N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X,—C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and—C(NR)NRR, where each X is independently a halogen (preferably —F or—Cl) and each R is independently hydrogen, alkyl, alkanyl, alkenyl,alkanyl, aryl, arylalkyl, arylaryl, heteroaryl, heteroarylalkyl orheteroaryl-heteroaryl, as defined herein. The actual substituentsubstituting any particular group will depend upon the identity of thegroup being substituted.

“Parent Xanthene Ring:” refers to a heteroaromatic ring system of a typetypically found in the xanthene class of fluorescent dyes (whichincludes rhodamine and fluorescein dyes, defined infra), i.e., aheteroaromatic ring system having the general structure:

In the parent xanthene ring depicted above, A¹ is —OH or —NH₂ and A² is═O or ═NH₂ ⁺. When A¹ is —OH and A² is ═O, the parent xanthene ring is afluorescein-type parent xanthene ring, which is defined in more detail,infra. When A¹ is —NH₂ and A² is ═NH₂ ⁺, the parent xanthene ring is arhodamine-type parent xanthene ring, which is defined in more detail,infra. When A¹ is —NH₂ and A² is ═O, the parent xanthene ring is arhodol-type parent xanthene ring. In the parent xanthene ring depictedabove, one or both nitrogens of A¹ and A² (when present) and/or one ormore of the carbon atoms at positions C1, C2, C4, C5, C7 and C8, can beindependently substituted with a wide variety of the same or differentsubstituents, as is well known in the art. Typical substituents include,but are not limited to, —X, —R, —OR, —SR, —NRR, perhalo (C₁-C₆) alkyl,—CX₃, —CF₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, —N₃, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O⁻,—C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR, where eachX is independently a halogen (preferably —F or —Cl) and each R isindependently hydrogen, (C₁-C₆) alkyl, (C₁-C₆) alkanyl, (C₁-C₆) alkenyl,(C₁-C₆) alkynyl, (C₅-C₂₀) aryl, (C₆-C₂₆) arylalkyl, (C₅-C₂₀) arylaryl,heteroaryl, 6-26 membered heteroarylalkyl or 5-20 memberedheteroaryl-heteroaryl. Moreover, the C1 and C2 substituents and/or theC7 and C8 substituents can be taken together to form substituted or=substituted (C₅-C₂₀) aryleno bridges. Generally, substituents groupswhich do not tend to quench the fluorescence of the parent xanthene ringare preferred, but in some embodiments quenching substituents may bedesirable. Substituents that tend to quench fluorescence of parentxanthene rings are electron-withdrawing groups, such as —NO₂, —F, —Br,—CN and —CF₃.

When A¹ is —NH₂ and/or A² is ═NH₂ ⁺, the xanthene nitrogens can beincluded in bridges involving the same nitrogen atom or adjacent carbonatoms, e.g., (C₁-C₁₂) alkyldiyl, (C₁-C₁₂) alkyleno, 2-12 memberedheteroalkyldiyl and/or 2-12 membered heteroalkyleno bridges.

Any of the substituents substituting carbons C1, C2, C4, C5, C7 or C8and/or the xanthene nitrogen atoms (when present) can be furthersubstituted with one or more of the same or different substituents,which are typically selected from the group consisting of —X, —R′, ═O,—OR′, —SR′, ═S, —NR′R′, ═NR′, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO,—NO₂, ═N₂, —N₃, —NHOH, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R′, —P(O)(O⁻)₂,—P(O)(OH)₂, —C(O)R′, —C(O)X, —C(S)R′, —C(S)X, —C(O)OR′, —C(O)O⁻,—C(S)OR′, —C(O)SR′, —C(S)SR′, —C(O)NR′R′, —C(S)NR′R′ and —C(NR)NR′R′,where each X is independently a halogen (preferably —F or —Cl) and eachR′ is independently hydrogen, (C₁-C₆) alkyl, 2-6 membered heteroalkyl,(C₅-C₁₄) aryl or heteroaryl.

Exemplary parent xanthene rings include, but are not limited to,rhodamine-type parent xanthene rings and fluorescein-type parentxanthene rings, each of which is defined in more detail, infra.

“Rhodamine-Type Parent Xanthene Ring:” refers to a parent xanthene ringin which A¹ is —NH, and A² is ═NH₂ ⁺, i.e., a parent xanthene ringhaving the general structure:

In the rhodamine-type parent xanthene ring depicted above, one or bothnitrogens and/or one or more of the carbons at positions C1, C2, C4, C5,C7 or C8 can be independently substituted with a wide variety of thesame or different substituents, as previously described for the parentxanthene rings. Exemplary rhodamine-type parent xanthene rings include,but are not limited to, the xanthene rings of the rhodamine dyesdescribed in U.S. Pat. No. 5,936,087; U.S. Pat. No. 5,750,409; U.S. Pat.No. 5,366,860; U.S. Pat. No. 5,231,191; U.S. Pat. No. 5,840,999; U.S.Pat. No. 5,847,162; U.S. application Ser. No. 09/277,793, filed Mar. 27,1999; PCT Publication WC) 97/36960; PCT Publication WO 99/27020; Saueret al., 1995, J. Fluorescence 5(3):247-261; Arden-Jacob, 1993, NeueLanwellige Xanthen-Farbstoffe für Fluoreszenzsonden and Farbstoff Laser,Verlag Shaker, Germany; and Lee et al., 1992, Nucl. Acids Res.20(10):2471-2483. Also included within the definition of “rhodamine-typeparent xanthene ring” are the extended-conjugation xanthene rings of theextended rhodamine dyes described in U.S. application Ser. No.09/325,243, filed Jun. 3, 1999.

“Fluorescein-Type Parent Xanthene Ring:” refers to a parent xanthenering in which A¹ is —OH and A² is ═O, i.e., a parent xanthene ringhaving the structure:

In the fluorescein-type parent xanthene ring depicted above, one or moreof the carbons at positions C1, C2, C4, C5, C7 or C8 can beindependently substituted with a wide variety of the same or differentsubstituents, as previously described for the parent xanthene rings.Exemplary fluorescein-type parent xanthene rings include, but are notlimited to, the xanthene rings of the fluorescein dyes described in U.S.Pat. No. 4,439,356; U.S. Pat. No. 4,481,136; U.S. Pat. No. 5,188,934;U.S. Pat. No. 5,654,442; U.S. Pat. No. 5,840,999; WO 99/16832; and EP 0050 684. Also included within the definition of “fluorescein-type parentxanthene ring” are the extended xanthene rings of the fluorescein dyesdescribed in U.S. Pat. No. 5,750,409 and U.S. Pat. No. 5,066,580.

“Xanthene Dye:” refers to a class of fluorescent dyes which consist of aparent xanthene ring substituted at the xanthene C-9 carbon with asubstituted phenyl ring or other, typically acyclic, substituent. Commonsubstituted phenyl rings found in xanthene dyes include, e.g.,2-carboxyphenyl, dihalo-2-carboxyphenyl, tetrahalo-2-carboxyphenyl,2-ethoxycarbonylphenyl, dihalo-2-ethoxycarbonylphenyl andtetrahalo-2-ethoxycarbonylphenyl. Common acyclic substituents found inxanthene dyes include, e.g., carboxyethyl and perfluoroalkyl (e.g.,trifluoromethyl, pentafluoroethyl and heptafluoropropyl). Typicalxanthene dyes include the fluorescein dyes and the rhodamine dyes, whichare described in more detail, infra.

“Rhodamine Dye:” refers to the subclass of xanthene dyes in which thexanthene ring is a rhodamine-type parent xanthene ring. Typicalrhodamine dyes include, but are not limited to, rhodamine B,5-carboxyrhodamine, rhodamine X (ROX), 4,7-dichlororhodamine X (dROX),rhodamine 6G (R6G), rhodamine 110 (R110), 4,7-dichlororhodamine 110(dR110), tetramethyl rhodamine (TAMPA) and4,7-dichloro-tetramethylrhodamine (dTAMRA). Additional typical rhodaminedyes can be found, for example, in U.S. Pat. No. 5,936,087; U.S. Pat.No. 5,750,409; U.S. Pat. No. 5,366,860; U.S. Pat. No. 5,231,191; U.S.Pat. No. 5,840,999; U.S. Pat. No. 5,847,162; U.S. application Ser. No.09/038,191, filed Mar. 10, 1998; U.S. application Ser. No. 09/277,793,filed Mar. 27, 1999; U.S. application Ser. No. 09/325,243, filed Jun. 3,1999; PCT Publication WO 97/36960; PCT Publication WO 99/27020; Sauer etal., 1995, J. Fluorescence 5(3):247-261; Arden-Jacob, 1993, NeueLanwellige Xanthen-Farbstoffe für Fluoreszerasonden and Farbstoff Laser,Verlag Shaker, Germany; and Lee et al., 1992, Nucl. Acids Res.20(10):2471-2483.

“Fluorescein Dye:” refers to the subclass of xanthene dyes in which theparent xanthene ring is a fluorescein-type parent xanthene ring. Typicalfluorescein dyes include, but are not limited to, 5-carboxyfluorescein(5-FAM), 6-carboxyfluorescein (6-FAM). Additional typical fluoresceindyes can be found, for example, in U.S. Pat. No. 5,750,409; U.S. Pat.No. 5,066,580; U.S. Pat. No. 4,439,356; U.S. Pat. No. 4,481,136; U.S.Pat. No. 5,188,934; U.S. Pat. No. 5,654,442; U.S. Pat. No. 5,840,999;PCT publication WO 99/16832; EP 0 050 684; and U.S. application Ser. No.08/942,067, filed Oct. 1, 1997.

5.3 The Rhodamine Dye Compounds

5.3.1 The Compounds Per Se

The rhodamine dyes of the invention are generally rhodamine-type parentxanthene rings substituted at the xanthene C9 position with a new bottomring. The new bottom ring is a phenyl group which bears fivesubstituents: an ortho carboxyl or sulfonate group (or salts thereof);one to three aminopyridinium (Pyr⁺) groups; and one alkylthio, arylthioor heteroarylthio group. The Pyr⁺ groups, which may be the same ordifferent, are typically the same and are attached to the new bottomring via the pyridinium ring nitrogen. The alkylthio, arylthio orheteroarylthio group is attached to the new bottom ring via the sulfuratom. As previously described in the Summary section, theaminopyridinium and/or alkylthio, arylthio or heteroarylthio groups maybe substituted or unsubstituted. The rhodamine dyes may also include anoptional linking moiety, described in more detail, infra.

Currently available red-emitting fluorescent dyes, such as rhodaminesand cyanines, suffer from undesirable water-solubility and/orphotostability characteristics. For example, due to their hydrophobicnature, most commercially-available rhodamine dyes are somewhatinsoluble in water. Red-emitting cyanine dyes such as Cy5, althoughwater-soluble, are photo unstable. Thus, available red-emittingrhodamine and cyanine dyes are not well-suited for many aqueous-basedbiological applications, such as cell staining or nucleic acidsequencing.

By virtue or their new bottom rings, the rhodamine dyes of the inventionovercome these limitations. While not intending to be bound by anyparticular theory, it is believed that the Pyr⁺ groups substituting thenew bottom ring render the dyes highly water-soluble. Quite importantly,rhodamine dyes substituted with these new bottom rings retain theircharacteristic photostability. Moreover, the new bottom ring tends toshift the emissions spectral properties of the dyes to the red by about5-30 nm, as compared with corresponding rhodamines dyes comprising aconventional bottom ring. Thus, the rhodamine dyes of the invention areideally suited for use as water-soluble laser dyes and in aqueous-basedbiological applications such as cell staining and nucleic acidsequencing. Quite significantly, when used to label terminators innucleic acid sequencing applications, the new rhodamine dyes of theinvention do not effect obscuring impurities which electrophoreticallymigrate in the range of DNA sequencing fragments. Obscuring impuritiesare commonly observed with conventionally-labeled terminators and aregenerally thought to be caused by unincorporated labelled-terminators.As a consequence, sequencing data obtained with terminators labeled withthe new rhodamine dyes of the invention is typically much higher inquality than that obtained with conventional terminators. Sequencingreactions obtained with terminators labeled with the new rhodamine dyesof the present invention may require less sample purification, or “cleanup” than those employing conventional terminators.

The rhodamine dyes of the invention are generally compounds according tostructural formula (I):

including any associated counterions, wherein:

-   -   E is carboxylic acid, sulfonic acid, or a salt thereof;    -   Y is a rhodamine-type parent xanthene ring connected to the        illustrated fully substituted phenyl ring at the C-9 carbon;    -   each Pyr⁺ is independently a substituted or unsubstituted        aminopyridinium group connected to the illustrated phenyl ring        via the pyridinium ring nitrogen;    -   S is sulfur,    -   n is one, two, or three; and    -   Z a substituted or unsubstituted (C₁-C₁₂)alkyl, (C₅-C₁₄)aryl or        heteroaryl.

The invention is based, in part, on the discovery that replacing thering or substituent attached to the xanthene C-9 carbon of conventionalrhodamine dyes with the new bottom rings described herein yieldsrhodamine dyes having superior water-solubility, photo stability and/orexcitation and emission spectral properties. As a consequence, those ofskill in the art will recognized that in the rhodamine dyes ofstructural formula (I), rhodamine-type parent xanthene ring Y can bederived from virtually any fluorescent rhodamine dye that is now knownor that will be later developed. Exemplary rhodamine-type parentxanthene rings that can comprise Y include, but are not limited to, anyof the substituted or unsubstituted xanthene rings of the rhodamine dyesdescribed in U.S. Pat. No. 5,936,087; U.S. Pat. No. 5,750,409; U.S. Pat.No. 5,366,860; U.S. Pat. No. 5,231,191; U.S. Pat. No. 5,840,999; U.S.Pat. No. 5,847,162; U.S. application Ser. No. 09/038,191, filed Mar. 10,1998; U.S. application Ser. No. 09/277,793, filed Mar. 27, 1999; U.S.application Ser. No. 09/325,243, filed Jun. 3, 1999; PCT Publication WO97/36960; PCT Publication WO 99/27020; Sauer at al., 1995, J.Fluorescence 5(3):247-261; Arden-Jacob, 1993, Neue LanwelligeWanthen-Farbstoffe für Fluoreszenzsonden and Farbstoff Laser, VerlagShaker, Germany, and Lee et al., 1992, Nucl. Acids Res.20(10):2471-2483, the disclosures of which are incorporated herein byreference.

While the dyes of structural formula (I) are useful in virtually anyaqueous-based applications employing red-emitting fluorescent dyes, suchas, for example, as water-soluble laser dyes, rhodamine-s according tostructural formula (I) which incorporate one or more optional linkingmoieties are particularly useful, as they can be specifically and/orpermanently conjugated to other compounds and/or substances so as tolabel the compounds or substances for subsequent detection. The linkingmoieties are attached to the rhodamine-type parent xanthene ring, eitherat a xanthene nitrogen, the xanthene C4 carbon, and/or the Zsubstituent. Thus, preferred rhodamine dyes of the invention arecompounds according to structural formula (I) in which the C4 carbonatom of Y, one or both nitrogen atoms of Y and/or substituent Z aresubstituted with a linking moiety of the formula -L-, wherein L is abond or linker. When the dye includes multiple linkers L, each may bethe same or different.

The nature of linker L will depend upon the particular application,point of attachment and type of conjugation desired. Linker L may beattached directly to the dye, or it may be spaced away from the dyethrough one or more intervening atoms that serve as a linker. In theformer embodiment, L represents a bond. In the latter embodiment, Lrepresents a linker of more than one atom. The linker can be hydrophilicor hydrophobic, long or short, rigid, semirigid or flexible, dependingupon the particular application. The linker can be optionallysubstituted with one or more substituents or one or more additionallinking groups, which may be the same or different, thereby providing a“polyvalent” linking moiety capable of conjugating with multiplemolecules or substances. Preferably, however, linker L does not includesuch additional substituents or linking groups.

A wide variety of linkers L comprised of stable bonds are known in theart, and include by way of example and not limitation, alkyldiyls,substituted alkyldiyls, alkylenos, substituted alkylenos,heteroalkyldiyls, substituted heteroalkyldiyls, heteroalkylenos,substituted heteroalkylenos, acyclic heteroatomic bridges, aryldiyls,substituted aryldiyls, arylaryldiyls, substituted atylaryldiyls,arylalkyldiyls, substituted arylalkyldiyls, heteroaryldiyls, substitutedheteroaryldiyls, heteroaryl-heteroaryldiyls, substitutedheteroaryl-heteroaryldiyls, heteroarylalkyldiyls, substitutedheteroarylalkyldiyls, heteroaryl-heteroalkyldiyls, substitutedheteroaryl-heteroalkyldiyls, and the like. Thus, linker L may includesingle, double, triple or aromatic carbon-carbon bonds,nitrogen-nitrogen bonds, carbon-nitrogen, carbon-oxygen bonds and/orcarbon-sulfur bonds, and may therefore include functionalities such ascarbonyls, ethers, thioethers, carboxamides, sulfonamides, ureas,urethanes, hydrazines, etc. In one embodiment, linker L has from 1-20non-hydrogen atoms selected from the group consisting of C, N, O, and Sand is composed of any combination of ether, thioether, amine, ester,carboxamide, sulfonamides, hydrazide, aromatic and heteroaromatic bonds.

Choosing a linker having properties suitable for a particularapplication is within the capabilities of those having skill in the art.For example, where a rigid linker is desired, L may be a rigidpolypeptide such as polyproline, a rigid polyunsaturated alkyldiyl or anaryldiyl, biaryldiyl, arylarydiyl, arylalkyldiyl, heteroaryldiyl,biheteroaryldiyl, heteroarylalkyldiyl, heteroaryl-heteroaryldiyl, etc.Where a flexible linker is desired, L may be a flexible polypeptide suchas polyglycine or a flexible saturated alkanyldiyl or heteroalkanyldiyl.Hydrophilic linkers may be, for example, polyalcohols or polyethers suchas polyalkyleneglycols. Hydrophobic linkers may be, for example,alkyldiyls or aryldiyls.

Linkers suitable for use in most biological applications include(C₁-C₁₂)alkyldiyls, particularly alkanylenos such as methano (—CH₂—),ethano (—CH₂—CH₂—), propano (—CH₂—CH₂—CH₂), butano (—CH₂—CH₂—CH₂—CH₂—),pentano (—CH₂—CH₂—CH₂—CH₂—CH₂) and hexano (—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—);(C₅-C₂₀) aryldiyls, particularly phena-1,3-diyl

and (C₆-C₂₆) arylalkyldiyls, particularly those having the structuralformula —(CHO₂)_(i)-φ- or —(CH₂)_(i)-Ψ-, where each i is independentlyan integer from 1 to 6, φ is phenyldiyl (especially phena-1,3-diyl orphena-1,4-diyl) and Ψ is naphthyldiyl (especially naphtha-2,6-diyl ornaphtha-2,7-diyl), Rigid linkers that are suitable for attaching thedyes of the invention to one another or to other dyes to createenergy-transfer dye pairs (described in more detail, infra) include—(CH₂)_(i)—NR″—C(O)-φ and —(CH₂)_(i)—NR″—C(O)-Ψ, where i, R″, φ and Ψare as previously defined. Particularly preferred are those linkers inwhich i is 1. Analogs of all of these linkers L containing one or moreheteroatomic groups, particularly those selected from the groupconsisting of O, S, N and NR″, where R″ is hydrogen or (C₁-C₆) alkyl,can also be conveniently used to space linkers from the rhodamine dyesof the invention. Linkers L tailored to specific applications arediscussed in more detail, infra.

Rhodamine dyes including a linking moiety can be conjugated to a varietyof different molecules and substances using a plethora of differentconjugation means. For example, the conjugation can be mediated viahydrophobic interactions, ionic attraction, through the use of pairs ofspecific binding molecules such as biotin and avidin/streptavidin orthrough covalent attachment. When conjugation via hydrophobicinteractions is desired, linker L is a hydrophobic moiety that iscapable of forming hydrophobic interactions with a hydrophobic moiety onthe molecule or substance to be conjugated. Typical hydrophobic moietiesinclude, but are not limited to, unsubstituted and substituted aryl,arylalkyl, arylaryl, heteroaryl, heteroarylaklyl andheteroaryl-heteroaryl groups. When the hydrophobic moiety issubstituted, the substituents are preferably nonpolar, more preferablyhydrophobic. Suitable hydrophobic moieties for forming non-covalentconjugates will be apparent to those of skill in the art.

When conjugation via ionic attraction is desired, L is a charged moietyhaving a net charge of a polarity opposite to a net charge on themolecule or substance to be conjugated. Typical charged moietiesinclude, by way of example and not limitation, quaternary ammoniums,carboxylates and sulfonates, including salts thereof. A variety ofcyclic quaternary ammoniums that are suitable for use in linkers aredescribed in U.S. Pat. No. 5,863,753 (see, e.g., Cols. 8-9), Thedisclosure of which is incorporated herein by reference.

When conjugation via pairs of specific binding molecules such as biotinand avidin/streptavidin is desired, L will constitute one member of thebinding pair. The molecule or substance to be conjugated will bear theother member of the binding pair. Where one of the members of thespecific binding pair is a small molecule, such as biotin or a hormone,that member preferably comprises L. A variety of biotins capable ofbeing covalently linked to reactive functional groups such as amines arecommercially available (e.g., Molecular Probes, Eugene, Oreg.). Thesebiotins can be incorporated into the dyes of the invention to yieldbiotin-labeled dyes suitable for non-covalent conjugation to a varietyof avidin/streptavidin-labeled molecules or substances.

Preferably, L is capable of mediating conjugation via covalentattachment. In this preferred embodiment, L bears a reactive functionalgroup (R_(x)). Covalent conjugates are obtained by reacting a rhodaminedye of the invention including a reactive group R_(x) with a molecule orsubstance that contains, or is modified to contain, one or morefunctional groups F_(x) that are complementary to reactive group R_(x).

The exact identities of R_(x) and F_(x) will depend upon the nature ofthe desired covalent linkage and the chemistry used to form the covalentlinkage. Generally, reactive group R_(x) is a functional group that iscapable of reacting with a complementary functional group F_(x) underspecified reaction conditions to form a covalent linkage. However, thoseof skill in the art will recognize that a variety of functional groupsthat are typically unreactive under certain reaction conditions can beactivated to become reactive. Groups that can be activated to becomereactive include, e.g., carboxylic acids and esters, including saltsthereof. Such groups are referred to herein as “activatable precursors”and are specifically intended to be included within the expression“reactive group.”

Pairs of reactive groups R_(x) and complementary groups F_(x) suitablefor forming covalent linkages with one another under a variety ofdifferent reaction conditions are well-known. Any of these complementarypairs of groups can be used to covalently conjugate the rhodamine dyesof the invention to other compounds or substances. In one convenientembodiment, reactive group R_(x) and complementary functional groupF_(x) comprise complementary electrophiles and nucleophiles (or theirrespective activatable precursors). In another convenient embodiment,reactive group R_(x) is a photoactivatable group that becomes chemicallyreactive only after illumination with light of an appropriate wavelengthand complementary functional group F_(x) is a group capable forming acovalent linkage with the chemically reactive species. Suchphotoactivatable groups can be conveniently used to photo cross-link therhodamine dyes of the invention to other molecules and/or substances.

As understood in the art, “activated esters” generally have the formula—C(O)Ω, where Ω is a good leaving group. Exemplary good leaving groupsinclude, by way of example and not limitation: oxysuccinimidyl;N-succinimidyl; oxysulfosuccinimidyl; 1-oxybenzotriazolyl; and —OR^(a),where R^(a) is selected from the group consisting of (C₄-C₂₀) cycloalkyl(e.g., cyclohexyl), heterocycloalkyl, (C₅-C₂₀) aryl, (C₅-C₂₀) arylsubstituted with one or more of the same or differentelectron-withdrawing groups (e.g., NO₂, —F, —Cl, —CN, —CF₃, etc.),heteroaryl, and heteroaryl substituted with one or more of the same ordifferent electron-withdrawing groups, n-dialkylaminoalkyls (e.g.,3-dimethylaminopropyl) and N-morpholinomethyl, or R^(a) is used to forman anhydride of the formula —OCOR^(b) or —OCNR^(b)NHR^(c), where R^(b)and R^(c) are each independently selected form the group consisting of(C₁-C₆) alkyl, (C₁-C₆) perhaloalkyl, (C₁-C₆) perfluoroalkyl and (C₁-C₆)alkoxy. A preferred activated ester is NHS ester.

Exemplary photoactivatable groups suitable for conjugation vialight-activated cross-linking include, but are not limited to, azido(—N₃), 4-azido-phenyl and 2-nitro-4-azido-phenyl. Conjugation usingphotoactivatable groups typically involves illuminating a mixturecomprising the photoactivatable dyes and the molecule or substance to beconjugated, followed by separation of unreacted dyes and byproducts.

As will be recognized by those of skill in the art, reactive group R_(x)can comprise any electrophilic, nucleophilic or photoactivatable groups.The selection of reactive group R_(x) used to covalently conjugate therhodamine dyes of the invention to the other molecule or substancetypically depends upon the identity of the complementary functionalgroup F_(x) on the molecule or substance to be conjugated. The types ofcomplementary functional groups typically present on molecules orsubstances to be conjugated include, but are not limited to, amines,thiols, alcohols, phenols, aldehydes, ketones, phosphates, imidazoles,hydrazines, hydroxylamines, mono- and disubstituted amines, halides,epoxides, sulfonate esters, carboxylic acids or carboxylates, or acombination of these groups. A single type of complementary functionalgroup may be available on the molecule or substance (which is typicalfor polysaccharides), or a variety of different complementary functionalgroups may be available (e.g. amines, thiols, alcohols, phenols), whichis typical for proteins. The molecule or substance may be conjugated tomore than one rhodamine dye, which may be the same or different.Although some selectivity can be obtained by carefully controlling thereaction conditions, selectivity of conjugation is best obtained byappropriate choice of reactive group in light of the availablecomplementary functional group(s) F_(x). In instances where the moleculeor substance to be conjugated does not contain available complementaryfunctional group(s) F_(x), it can be modified to contain such groupsusing any of a variety of standard techniques.

In a preferred embodiment, reactive group R_(x) is a group that reactswith, or that can be readily activated to react with, an amine, a thiolor an alcohol. In a particularly preferred embodiment, one of reactivegroup R_(x) or complementary functional group F_(r) is a carboxylic acid(or a salt thereof) or an activated ester, most preferably aN-hydroxysuccinimidyl (NHS) ester, and the other is an amine, preferablya primary amine. The NHS ester may be conveniently obtained by reactinga rhodamine dye of the invention including a carboxylic acid reactivegroup R_(x) with N-hydroxysuccinimide in the presence of an activatingagent (e.g., dicyclohexylcarbodiimide) according to known methods.

For a discussion of the various reactive groups R_(x) and respectivecomplementary functional groups F_(x) that can be conveniently used tocovalently conjugate the rhodamine dyes of the invention to a variety ofbiological and other molecules or substances, as well as reactionconditions under which the conjugation reactions can be carried out, seeHaugland, 1996, Molecular Probes Handbook of Fluorescent Probes andResearch Chemicals, Molecular Probes, Inc.; Brinkley, 1992, BioconjugateChem. 3:2 and Garman, 1997, Non-Radioactive Labelling: A PracticalApproach, Academic Press, London, as well as the references cited in allof the above.

In one illustrative embodiment, rhodamine dyes of the invention whichare capable of being covalently conjugated to other compounds and/orsubstances are compounds according to structural formula (Ia):

including any associated counter ions, wherein:

-   -   E, Y, Pyr⁺ and S are as previously defined for structural        formula (I);    -   Z¹ is a substituted or unsubstituted (C₁-C₁₂) alkyldiyl,        (C₅-C₁₄) or heteroaryldiyl;    -   L is a bond or a linker as previously described;    -   R_(x) is a reactive group as previously described; and    -   n is one, two, or three.

In another illustrative embodiment, rhodamine dyes of the inventionwhich are capable of being covalently conjugated to other compoundsand/or substances are compounds according to structural formula (Ib):

including any associated counterions, wherein:

-   -   E, Y, Pyr⁺, S and Z are as previously defined for structural        formula (I);    -   L and R_(x) are as previously defined for structural formula        (Ia), where L is connected to the C4 carbon atom or a nitrogen        atom of Y; and    -   n is one, two, or three.

While not intending to be bound by any particular theory, it is believedthat the Pyr⁺ groups on the new bottom rings in the compounds ofstructural formulae (I), (Ia) and (Ib) account for the water-solubilityof the rhodamine dyes of the invention.

As will be illustrated more thoroughly below, the Pyr⁺ groups areintroduced into the rhodamine dyes of the invention by displacingfluorine atoms of the corresponding tetrafluororhodamine precursor withthe desired aminopyridine reactant, e.g. Scheme (I), using a reactionsimilar to that described in Weiss, R. et al, 1995, Angew. Chem. Int.Ed. Engl. 34:1319-21; and Koch, A. et al, 1993, Jour. Org. Chem.58:1409-14. As a consequence, unless mixtures of different aminopyridinereactants are used in the displacement reaction, all of the Pyr⁺ groupson the new bottom phenyl rings are identical. According to the reaction,the Pyr⁺ groups are attached to the new bottom phenyl ring via thepyridinium ring nitrogen.

The pyridinium ring carbons may be independently substituted with a widevariety of the same or different substituents. The desired substituentsare introduced as substituents on the aminopyridine reactants. Thesecarbons may be substituted with virtually any group, with one caveat: inorder to avoid deleteriously affecting the displacement reaction, thepyridine ring carbons should not be directly substituted withelectron-withdrawing groups, e.g., —F, —Cl, —CF₃, —NO₂, —CN, —N₃, etc.However, such electron-withdrawing groups can be included on thesubstituents, as long as it is not attached directly to a pyridine ringcarbon. For example, while a pyridine ring carbon should not be directlysubstituted with —F, —Cl or —CF₃, it may be substituted with other, lesselectronegative or electron-withdrawing, haloalkyls (e.g., —CH₂—CH₂F).Identifying substituents which are suitably non-electron-withdrawing iswithin the capabilities of those having skill in the art. Typical groupsuseful for substituting the pyridinium ring carbons include, but are notlimited to, —R, —OR, —SR, —NRR, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, —C(O)R,—C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR,—C(O)NRR, —C(S)NRR and —C(NR)NRR, where each R is independentlyhydrogen, (C₁-C₆) alkyl or heteroalkyl, (C₅-C₁₄) aryl or heteroaryl. TheR groups may be further substituted with one or more of the same ordifferent substituents, which are typically selected from the groupconsisting of —X, —R′, ═O, —OR′, —SR′, ═S, —NR′R′, ═NR′, —CX₃, —CN,—OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O′, —S(O)₂OH,—S(O)₂R′, —C(O)R′, —C(O)X, —C(S)R′, —C(S)X, —C(O)OR′, —C(O)O⁻, —C(S)OR′,—C(O)SR′, —C(S)SR′, —C(O)NR′R′, —C(S)NR′R′ and —C(NR)NR′R′, where each Xis independently a halogen (preferably —F or —Cl) and each R′ isindependently hydrogen, (C₁-C₆) alkyl, 2-6 membered heteroalkyl,(C₅-C₁₄) aryl or heteroaryl. Preferably, the pyridinium ring carbons areunsubstituted. When substituted, the most preferred substituents are thesame or different (C₁-C₆) alkyls. Preferably, the pyridinium carbons areunsubstituted. When substituted, the substituents are preferably thesame or different (C₁-C₆) alkyls.

The amino groups of the Pyr⁺ substituents may be a primary, secondary ortertiary amino group, but is typically a tertiary amino. The nitrogensubstituents, R, are typically the same or different (C₁-C₆) alkyl orheteroalkyl. The alkyl or heteroalkyl can be further substituted withone or more of the same or different groups, as previously described forR, above.

Alternatively, the nitrogen atom may be included in a ring structurehaving from 2 to 5 ring atoms. The ring may contain, in addition to theamino nitrogen atom, one or more of the same or different heteroatoms,which are typically selected from the group consisting of O, S and N.The ring atoms can be further substituted with any of the previouslydescribed substituent groups. Preferably, the amino group isdimethylamino or morpholino.

In the compounds of structural formulae (I), (Ia) and (Ib), the Z orsubstituent may also be substituted or unsubstituted, but is preferablyunsubstituted. When Z is an alkyl or Z¹ is an aryldiyl, virtually anygroup can be used to substitute Z or Z¹. Typical substituents include,but are not limited to, —X, —R, ═O, —OR, —SR, ═S, —NRR, ═NR, —CX₃, —CN,—OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH,—S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O⁻, —C(S)OR,—C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR, where each X isindependently a halogen (preferably —F or —Cl) and each R isindependently hydrogen, (C₁-C₆) alkyl, 2-6 membered heteroalkyl,(C₅-C₁₄) aryl or heteroaryl. The R groups may be further substitutedwith one or more of the same or different substituents, which aretypically selected from the group consisting of —X, —R′, ═O, —OR′, —SR′,═S, —NR′R′, ═NR′, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂,—N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R′, —C(O)R′, —C(O)X, —C(S)R′, —C(S)X,—C(O)OR′, —C(O)O⁻, —C(S)OR′, —C(O)SR′, —C(S)SR′, —C(O)NR′R′, —C(S)NRR′and —C(NR)NR′R′, where each X is independently a halogen (preferably —For —Cl) and each R′ is independently hydrogen, (C₁-C₆) alkyl, 2-6membered heteroalkyl, (C₅-C₁₄) aryl or heteroaryl.

However, referring to Scheme (I), it has been observed that thedisplacement reaction does not proceed efficiently with arylthio andheteroarylthio compounds 108 that are substituted at the aromatic ringatoms with halogen groups. Thus, when Z is an aryl or heteroaryl and/orZ′ is an aryldiyl or heteroaryldiyl, the aromatic ring atoms should notbe substituted directly with halogens. However, as described above forthe pyridinium ring carbon substituents, the halogens can be included ina substituent (e.g., a haloalkyl), so long as the substituent is notelectronegative to disrupt the displacement reaction. Thus, with theexception of halogen, groups to substitute aryl, heteroaryl, aryldiyland heteroaryldiyl Z and Z¹ groups are typically any of the groupsdescribed above for when Z is an alkyl or Z¹ is an alkyldiyl.

The rhodamine dyes of the invention will now be more fully described byreference to various preferred embodiments. In one preferred embodiment,the rhodamine dyes of the invention are compounds according tostructural formulae (I), (Ia) and (Ib) in which each Pyr⁺ is the sameand has the structure:

including any associated counter ions, wherein:

-   -   R²² is selected from the group consisting of hydrogen and        (C₁-C₆) alkyl;    -   R²³ is selected from the group consisting of hydrogen and        (C₁-C₆) alkyl;    -   R²⁴, when taken alone, is selected from the group consisting of        (C₁-C₆) alkyl, or when taken together with R²⁶ is (C₄-C₁₀)        alkyldiyl, (C₄-C₆) alkyleno, heteroalkyldiyl or heteroalkyleno;    -   R^(24′), when taken alone, is selected from the group consisting        of (C₁-C₆) alkyl, or when taken together with R²⁴ is (C₄-C₁₀)        alkyldiyl, (C₄-C₆) alkyleno, heteroalkyldiyl or heteroalkyleno;    -   R²⁵ is selected from the group consisting of hydrogen and        (C₁-C₆) alkyl; and    -   R²⁶ is selected from the group consisting of hydrogen and        (C₁-C₆) alkyl.

In the Pyr⁺ substituents of structural formula (P.1), the dashed line atthe pyridinium ring nitrogen indicates the point of attachment to thephenyl ring in the compounds of structural formulae (I), (Ia) and/or(D). Preferred Pyr⁺ substituents according to structural formula (P.1)are those in which R²², R²³, R²⁵ and R²⁶ are each hydrogen and/or inwhich R²⁴ and R^(24′), when taken alone, are each the same (C₁-C₆) alkylor, when taken together are a 4-6 membered heteroalkyleno having asingle oxygen heteroatom. Particularly preferred Pyr⁺ substituentsaccording to structural formula (P.1) are 4-(dimethylamino)pyridiniumand 4-(morpholino)pyridinium.

In another preferred embodiment, the rhodamine dyes of the invention arecompounds according to structural formulae (I) and (Ib) in which Z isselected from the group consisting of (C₁-C₁₂) alkyl, (C₁-C₁₂) alkylsubstituted with one or more of the same or different W¹ groups;(C₅-C₁₄) aryl, (C₅-C₁₄) aryl substituted with one or more of the same ordifferent W² groups, 5-14 membered heteroaryl or heteroarylindependently substituted with one or more of the same or different W²groups, wherein:

-   -   each W¹ is independently selected from the group consisting of        —X, —R, ═O, —OR, —SR, ═S, —NRR, ═NR, —CX₃, —CN, —OCN, —SCN,        —NCO, —NCS, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R,        —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O⁻, —C(S)OR,        —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR, where each X        is independently a halogen (preferably —F or —Cl) and each R is        independently hydrogen or (C₁-C₆) alkyl; and    -   each W² is independently selected from the group consisting of        —R, —OR, —SR, —NRR, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO,        —NO₂, —N₃, —S(O)₂O⁻—, —S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R,        —C(S)X, —C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR,        —C(S)NRR and —C(NR)NRR, R is as previously defined for W¹.

Preferably, Z is unsubstituted. However, when Z is a substituted(C₅-C₁₄) aryl or a substituted heteroaryl, the most preferredsubstituents are those that are not electron-withdrawing. The mostpreferred heteroaryl groups, whether substituted or unsubstituted, arethose in which any heteroatoms are nitrogens. Especially preferredamongst these preferred heteroaryls are pyridinyl and purinyl. The mostpreferred aryl groups, whether substituted or unsubstituted are phenyland naphthyl.

In another preferred embodiment, the rhodamine dyes of the invention arecompounds according to structural formula (Ia) in which Z¹ is selectedfrom the group consisting of (C₁-C₁₂) alkyldiyl, (C₁-C₁₂) alkyldiylsubstituted with one or more of the same or different W¹ groups;(C₅-C₁₄) aryldiyl, (C₅-C₁₄) aryldiyl substituted with one or more of thesame or different W² groups, 5-14 membered heteroaryldiyl orheteroaryldiyl independently substituted with one or more of the same ordifferent W² groups, wherein W¹ and W² are as defined above.

Preferably, Z¹ is unsubstituted. However, when Z¹ is a substituted(C₅-C₁₄) aryldiyl or a substituted heteroaryldiyl, the most preferredsubstituents are those that are not electron-withdrawing. The mostpreferred heteroaryldiyl groups, whether substituted or unsubstituted,are those in which any heteroatoms are nitrogens. Especially preferredamongst these preferred heteroaryldiyls are pyridindiyl and purindiyl.The most preferred aryldiyl groups, whether substituted or unsubstitutedare phendiyl and naphthadiyl, especially phena-1,3-diyl, phena-1,4-diyl,naphtha-2,6-diyl and naphtha-2,7-diyl.

In yet another preferred embodiment, the rhodamine dyes of the inventionare compounds according to structural formula (Ia) in whichrhodamine-type parent xanthene ring is a compound according tostructural formula (Y-1):

including any associated counterions, wherein:

R¹ and R² are each independently selected from the group consisting ofhydrogen and (C₁-C₆) alkyl;

R³, when taken alone, is selected from the group consisting of hydrogen,(C₁-C₆) alkyl, (C₅-C₁₄) aryl and (C₅-C₁₄) arylaryl, or when takentogether with R^(3′) is (C₄-C₆) alkyldiyl or (C₄-C₆) alkyleno, or whentaken together with R² or R⁴ is (C₂-C₆) alkyldiyl or (C₂-C₆) alkyleno;

R^(3′), when taken alone, is selected from the group consisting ofhydrogen, (C₁-C₆) alkyl, (C₅-C₁₄) aryl and (C₅-C₁₄) arylaryl, or whentaken together with R³ is (C₄-C₆) alkyldiyl or (C₄-C₆) alkyleno, or whentaken together with R² or R⁴ is (C₂-C₆) alkyldiyl or (C₂-C₆) alkyleno;

R⁴, when taken alone, is selected from the group consisting of hydrogenand (C₁-C₆) alkyl, or when taken together with R³ or R^(3′) is (C₂-C₆)alkyldiyl or (C₂-C₆) alkyleno;

R⁵, when taken alone, is selected from the group consisting of hydrogenand (C₁-C₆) alkyl, or when taken together with R⁶ or R^(6′) is (C₂-C₆)alkyldiyl or (C₂-C₆) alkyleno;

R⁶, when taken alone, is selected from the group consisting of hydrogen,(C₁-C₆) alkyl, (C₅-C₁₄) aryl and (C₅-C₁₄) arylaryl, or when takentogether with R⁶ is (C₄-C₆) alkyldiyl or (C₄-C₆) alkyleno, or when takentogether with R⁵ or R⁷ is (C₂-C₆) alkyldiyl or (C₂-C₆) alkyleno;

R^(6′), when taken alone, is selected from the group consisting ofhydrogen, (C₁-C₆) alkyl, (C₅-C₁₄) aryl and (C₅-C₁₄) arylaryl, or whentaken together with R⁶ is (C₄-C₆) alkyldiyl or (C₄-C₆) alkyleno, or whentaken together with R⁵ or R⁷ is (C₂-C₆) alkyldiyl or (C₂-C₆) alkyleno;

R⁷, when taken alone, is selected from the group consisting of hydrogenand (C₁-C₆) alkyl, or when taken together with R⁶ or R^(6′) is (C₂-C₆)alkyldiyl or (C₂-C₆) alkyleno;

R⁸, when taken alone, is selected from the group consisting of hydrogenand (C₁-C₆) alkyl; and

R⁹ indicates the point of attachment to phenyl bottom ring.

In another preferred embodiment, the rhodamine dyes of the invention arecompounds according to structural formula (Ia) in which rhodamine-typeparent xanthene ring is a compound according to structural formula(Y-2), (Y-3) or (Y-4):

including any associated counterions, wherein:

-   -   R¹, R³, R⁴, R⁵, R^(6′), R⁸ and R⁹ are as previously defined for        structural formula (Y-1); and    -   R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹        are each independently selected from the group consisting of        hydrogen and (C₁-C₆) alkyl, or R¹⁰, R¹¹, R¹² and R¹³ taken        together are (C₅-C₁₄) aryleno or (C₅-C₁₄) aryleno substituted        with one or more of the same or different (C₁-C₆) alkyl, or R¹⁸,        R¹⁹, R²⁰ and R²¹ taken together are (C₅-C₁₄) aryleno or (C₅-C₁₄)        aryleno substituted with one or more of the same or different        (C₁-C₆) alkyl.

The dashed bonds in structural formulae (Y-2) and (Y-4) indicate bondswhich can each, independently of one another, be a single or a doublebond. When these bonds are double bonds, one of R¹⁰ or R¹¹ and one ofR¹² or R¹³ are taken together to form a bond and one of R¹⁸ or R¹⁹ andone of R²⁰ or R²¹ are taken together to form a bond. When these bond aresingle bonds, the R¹⁰, R¹¹, R¹², R¹³, R¹⁸, R¹⁹, R²⁰ and R²¹ substituentsare as defined above.

In still another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (Ib) in whichrhodamine-type parent xanthene ring Y is selected from the groupconsisting of (Y-1), (Y-2), (Y-3) and (Y-4), where R¹, R², R³, R⁵, R⁶,R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ andR²¹ are as previously defined, and either R^(3′) or R⁴ indicates thepoint of attachment of substituent L. When substituent L is attached toR^(3′), R⁴ is as previously defined. When substitutent L is attached toR⁴, R^(3′) is as previously defined.

In still another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formulae (Ia) and (Ib)in which Y is selected from the group consisting of (Y-1), (Y-2), (Y-3)and (Y-4), and further in which:

-   -   any alkyl groups are alkanyls selected from the group consisting        of methanyl, ethanyl and propanyl;    -   any aryl groups are phenyl or naphthyl;    -   any arylaryl groups are biphenyl;    -   any alkyldiyl or alkyleno bridges formed by taking R² together        with R³ or R^(3′) are alkanyldiyls or alkanos, especially those        selected from the group consisting of ethano, propano,        1,1-dimethylethano, 1,1-dimethylpropano and        1,1,3-trimethylpropano;    -   any alkyldiyl or alkyleno bridges formed by taking R³ together        with R^(3′) are alkanyldiyl or allcano, especially butano;    -   any alkyldiyl or alkyleno bridges formed by taking R⁴ together        with and R³ or R^(3′) are alkanyldiyls or alkanos, especially        those selected from the group consisting of ethano, propano,        1,1-dimethylethano, 1,1-dimethylpropano and        1,1,3-trimethylpropano;    -   any alkyldiyl or alkyleno bridges formed by taking R⁵ together        with R⁶ or R^(6′) are alkanyldiyls or alkanos, especially those        selected from the group consisting of ethano, propano,        1,1-dimethylethano, 1,1-dimethylpropano and        1,1,3-trimethylpropano;    -   any alkyldiyl or alkyleno bridges formed by taking R⁶ together        with R^(6′) are alkanyldiyl or alkano, especially butano;    -   any alkyldiyl or alkyleno bridges formed by taking R⁷ together        with R⁶ or R^(6′) are alkanyldiyls or alkanos, especially those        selected from the group consisting of ethano, propano,        1,1-dimethylethano, 1,1-dimethylpropano and        1,1,3-trimethylpropano;    -   any aryleno bridges formed by taking R¹⁰, R¹¹, R¹² and R¹³        together are benzo; and    -   any aryleno bridges formed by taking R¹⁸, R¹⁹, R²⁰ and R²¹        together are benzo.

In yet another preferred embodiment, the rhodamine dyes of the inventionare compounds according to structural formula (Ia) in whichrhodamine-type parent xanthene ring Y is selected from the followinggroup of compounds, where R⁹ is as previously defined for structuralformula (Y-1):

In yet another preferred embodiment, the rhodamine dyes of the inventionare compounds according to structural formula (Ia) in which:

-   -   Y is selected from the group consisting of (Y-1), (Y-2), (Y-3),        (Y-4), (Y-20a), (Y-21a), (Y-22a), (Y-23a), (Y-24a), (Y-25a),        (Y-31a), (Y-34a), (Y-35a), (Y-36a), (Y-39a), (Y-41a), (Y-42a),        (Y-43a), (Y-44a), (Y-45a), and (Y-46a);    -   Z¹ is selected from the group consisting of (C₁-C₁₂) alkyleno,        (C₁-C₁₂) alkano, (C₅-C₁₀) aryldiyl, phenyldiyl, phena-1,4-diyl,        naphthadiyl, naphtha-2,6-diyl and heteroaryldiyl, pyridindiyl        and purindiyl.

In yet another preferred embodiment, the rhodamine dyes of the inventionare compounds according to structural formula (Ia) in which:

-   -   L is a bond; and    -   Y is selected from the group consisting of (Y-1), (Y-2), (Y-3),        (Y-4), (Y-20a), (Y-21a), (Y-22a), (Y-23a), (Y-24a), (Y-25a),        (Y-31a), (Y-34a), (Y-35a), (Y-36a), (Y-39a), (Y-41a), (Y-42a),        (Y-43a), (Y-44a), (Y-45a), and (Y-46a);

In still another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (Ia) in which:

-   -   R_(x) is any of the electrophilic or nucleophilic groups; and    -   Y is selected from the group consisting of (Y-1), (Y-2), (Y-3),        (Y-4), (Y-20a), (Y-21a), (Y-22a), (Y-23a), (Y-24a), (Y-25a),        (Y-31a), (Y-34a), (Y-35a), (Y-36a), (Y-39a), (Y-41a), (Y-42a),        (Y-43a), (Y-44a), (Y-45a), and (Y-46a);

In yet another preferred embodiment, the rhodamine dyes of the inventionare compounds according to structural formula (Ib) in which Y isselected from the group consisting of (Y-1), (Y-2), (Y-3), (Y-4),(Y-20a), (Y-21a), (Y-22a), (Y-23a), (Y-24a), (Y-25a), (Y-31a), (Y-34a),(Y-35a), (Y-36a), (Y-39a), (Y-41a), (Y-43a) and (Y-46a), wheresubstituent L is attached to the xanthene C4 carbon. In structures(Y-23a), (Y-24a) and (Y-25a), the C4 carbon in the illustrated tautomeris bridged to the xanthene nitrogen. Those of skill in the art willrecognize that due to tautomerism, the carbons labeled C4 and C5 (in thedefinition of rhodamine-type parent xanthene ring) are essentiallyequivalent. Thus, in the illustrated structures of compounds (Y-23a),(Y-24a) and (Y-25a), substituent L is bonded to the C5 carbon.Particularly preferred compounds according to this aspect of theinvention are those compounds in which:

-   -   Z is selected from the group consisting of (C₁-C₁₂) alkyl,        (C₁-C₁₂) alkanyl, (C₅-C₁₀) aryl, phenyl, naphthyl, naphth-1-yl,        naphth-2-yl, pyridyl and purinyl; and/or    -   L is selected from the group consisting of (C₁-C₆) alkyldiyl,        (C₁-C₆) alkano, (C₅-C₂₀) aryldiyl, phenyldiyl, phena-1,4-diyl,        naphthyldiyl, naphtha-2,6-diyl, naphtha-2,7-diyl, (C₆-C₂₆)        arylalkyldiyl-(CH₂)_(i)-φ-, —(CH₂)_(i)-Ψ-,        —(CH₂)_(i)—NHR″—C(O)-φ- and —(CH₂)_(i)—NHR″—C(O)-Ψ-, where each        i is independently an integer from 1 to 6, R″ is hydrogen or        (C₁-C₆) alkyl, φ is (C₅-C₂₀) aryldiyl, phenyldiyl or        phena-1,4-diyl and Ψ is naphthyldiyl, naphtha-2,6-diyl or        naphtha-2,7-diyl; and/or    -   R_(x) is any electrophilic or nucleophilic group.

In yet another preferred embodiment, the rhodamine dyes of the inventionare compounds according to structural formula (Ib) in which Y isselected from the group consisting of (Y-1), (Y-2), (Y-3), (Y-4), wheresubstituent L is attached to R³, or one of the following group ofcompounds, where R⁹ is as previously defined for structural formula(Y-1) and the dashed line at the nitrogen atom indicates the point ofattachment of substituent L:

In still another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (Ib) in which:

-   -   Y is selected from the group consisting of (Y-1), (Y-2), (Y-3),        (Y-4), (Y-20b), (Y-21b), (Y-22b), (Y-23b), (Y-24b), (Y-25b),        (Y-31b), (Y-34b), (Y-35b), (Y-36b), (Y-39b), (Y-42b), (Y-43b),        and (Y-46b); and    -   Z is selected from the group consisting of (C₁-C₁₂) alkyl,        (C₁-C₁₂) alkanyl, (C₅-C₁₀) aryl, phenyl, naphthyl, naphth-1-yl,        naphth-2-yl, pyridyl and purinyl, where in structures (Y-1)        through (Y-4) L is attached to R^(3′).    -   L is selected from the group consisting of (C₁-C₆) alkyldiyl,        (C₁-C₆) alkano, (C₅-C₂₀) aryldiyl, phenyldiyl, phena-1,4-diyl,        naphthyldiyl, naphtha-2,6-diyl, naphtha-2,7-diyl, (C₆-C₂₆)        arylalkyldiyl —(CH₂)_(i)-φ-, —(CH₂)_(i)-Ψ-,        —(CH₂)_(i)—NR″—C(O)-φ- and —(CH₂)_(i)—NR″—C(O)-Ψ-, where each i        is independently an integer from 1 to 6, R″ is hydrogen or        (C₁-C₆) alkyl, φ is (C₅-C₂₀) aryldiyl, phenyldiyl or        phena-1,4-diyl and Ψ is naphthyldiyl, naphtha-2,6-diyl or        naphtha-2,7-diyl, and where in structures (Y-1) through (Y-4), L        is attached to R^(3′).

R_(x) is any electrophilic or nucleophilic group.

In still another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formulae (I), (Ia) and(Ib), including any of their respective previously-described preferredembodiments, in which E is a carboxyl or a salt thereof.

In the rhodamine dyes of the invention, aside from substituent E, theexact positions of the various substituents substituting the new bottomring, i.e., the Pyr⁺ and —S—Z¹-L-R_(x) substituents illustrated instructural formula (Ia) and the Pyr⁺ and —S—Z substituents illustratedin structural formula (Ib) in the dyes of the present invention may beunknown. However, it has been confirmed by nuclear magnetic resonance(NMR) that the bottom ring contains one, two, or three Pyr⁺substituents. Based upon the starting materials used to synthesize therhodamine dyes of the invention and the nature of the synthesisreactions (discussed in more detail in Section 5.3.2, infra), it isbelieved that the various rhodamine dyes according to structural formula(I) have the regiochemistry depicted in structural formula (Ic)(illustrated with Pyr⁺=4-(dimethylamino)pyridinium and E=—CO₂H):

It will be understood, however, that the Applicants do not intend to bebound by any particular structural representation, and that theinvention is intended to encompass the rhodamine dyes that are obtainedby the synthetic methods described herein, regardless of their specificregiochemistry.

In addition to the above-described structural isomerism about the newbottom ring, those of skill in the art will appreciate that many of thecompounds encompassed by formulae (I), (Ia) and/or (Ib) as describedherein, as well as many of the energy-transfer dye pairs and otherconjugates described infra, may exhibit the phenomena of tautomerism,conformational isomerism, geometric isomerism and/or stereoisomerism. Asthe formulae drawings within this specification can represent only oneof the possible tautomeric, conformational isomeric, enantiomeric orgeometric isomeric forms, it should be understood that the inventionencompasses any tautomeric, conformational isomeric, enantiomeric and/orgeometric isomeric forms of the compounds having one or more of theutilities described herein.

Moreover, as the compounds of the invention may bear one or morepositive charges or negative charges, depending upon their physicalstate, they may be in the form of a salts or may have counterionsassociated therewith. The identity(ies) of any associated counterions istypically dictated by the synthesis and/or isolation methods by whichthe compounds are obtained. Typical counterions include, but are notlimited to, halides, acetate, trifluoroacetate, etc. and mixturesthereof. It will be understood that the identity(ies) of any associatedcounterions is not a critical feature of the invention, and that theinvention encompasses any type of associated counterion. Moreover, asthe compounds can exists in a variety of different forms, the inventionis intended to encompass not only forms that are in association withcounterions (e.g., dry salts), but also forms that are not inassociation with counterions (e.g., aqueous solutions).

5.3.2 Methods of Synthesis

The rhodamine dyes of the invention, and in particular rhodamine dyesaccording to structural formula (Ia) in which each Pyr⁺ is4-(dimethylamino)pyridinium can be readily synthesized fromcorresponding tetrafluoro-rhodamine starting materials according toScheme I, below. Although a specific regiochemistry is depicted inScheme I, it will be understood that the structural representations axeillustrative only. In Scheme I, Y, S, L, Z¹ and R_(x) are as previouslydefined for structural formula (Ia). E is illustratively a carboxyl, butcould be a sulfonate.

Referring to Scheme I, 4-(dimethyl)aminopyridine is added to a solutionof tetrafluoro-rhodamine 100. The reaction is followed by thin-layerchromatography (TLC). The compound will depend upon the identity ofrhodamine-type parent xanthene ring Y. Once the reaction has gone tocompletion, thiol 108 is added to the mixture and the reaction monitoredwith TLC. Dye 110 is purified by reverse phase chromatography or otherstandard methods. Compounds in which Pyr⁺ is other than4-(dimethylamino)pyridinium are prepared in an analogous manner from theappropriate aminopyridine starting material. Compounds havingnon-identical Pyr⁺ groups may be prepared using a mixture of theappropriate aminopyridines.

Compounds according to structural formula (Ib) are synthesized in asimilar manner from the appropriate tetrafluoro-rhodamine derivative 101(illustrated below) using thiol Z—SH (106), where Z is as defined instructural formula (Ib), to displace a DMAP⁺ group:

In derivative 101, Y, L and R_(x) are as previously defined forstructural formula (Ib). When linking moiety -L-R_(x) is attached at axanthene nitrogen, tetrafluoro-rhodamine 101 is prepared as illustratedin Scheme I. When linking moiety is attached at the xanthene C4position, tetrafluoro-rhodamine 101 is prepared from a substitutedmonomer 118 (R⁴=CH₂X or L-R_(x)). The full range of rhodamine dyesdescribed herein can be synthesized by routine modification of thesemethods. Where necessary, any reactive functionalities on linker L, e.g.reactive functional group R_(x) can be protected using known groups andmethods (see, e.g., Greene & Wuts, Protective Groups in OrganicChemistry, 1991, 2^(nd) Edition, John Wiley & Sons, NY). Dyes in whichR_(x) is a carboxylic acid or a salt thereof are particularlyadvantageous, as the carboxyl group does not require protection duringsynthesis.

The appropriate tetrafluoro-rhodamine starting materials can be producedin the usual manner for the synthesis of rhodamines by condensing 1 molof an appropriate 3-aminophenol derivative with 1 mol oftetrafluorophthalic anhydride 114 (Aldrich Chemical Co, St. Louis, Mo.)according to known techniques (see, e.g., U.S. Pat. No. 5,750,409;Römpps' Chemie Lexicon, 8^(th) Edition, pp. 3580). The appropriate3-aminophenol starting materials are either commercially available orcan be readily obtained using standard synthesis methods. Arepresentative synthesis for preparing tetrafluororhodamine startingmaterial 100 in which rhodamine-type parent xanthene ring Y is acompound according to structural formula (Y-1) (compound 120) isoutlined in Scheme II.

In Scheme TI, the various R^(n) substituents are as previously definedfor structural formula (Y-1). According to Scheme II, 1 mol of3-aminophenol derivative 112 is refluxed with 1 mol oftetrafluorophthalic anhydride 114 (Aldrich) to yield intermediate 116.Intermediate 116 is refluxed with 3-aminophenol derivative 118 to yieldtetrafluoro-rhodamine 120. Depending upon the identity of rhodamine-typeparent xanthene ring Y, the tetrafluoro-rhodamine 120 is isolated byreverse phase or normal phase column chromatography.

An analogous method for synthesizing tetrafluoro-rhodamine 100 in whichrhodamine-type parent xanthene ring Y is a compound according tostructural formula (Y-2) is illustrated in Scheme III:

In Scheme III, the various R″ substituents are as previously defined forstructural formula (Y-2). Dyes according to structural formula (Ia) inwhich the rhodamine-type parent xanthene ring is a compound according tostructural formula (Y-3) or (Y-4) are prepared in an analogous mannerfrom the appropriate starting materials.

Methods of preparing tetrafluoro-rhodamine derivative 101 in which Y isa compound according to structural formula (Y-1) or (Y-2) in which thelinking moiety is attached to the xanthene nitrogen (i.e., at positionR^(3′)) are illustrated in Schemes IIb and IIIb, respectively:

The various aminophenol starting materials are either commerciallyavailable or can be prepared using routine methods. Exemplary synthesesare provided in the Examples section. The full scope of the rhodaminecompounds described herein can be readily synthesized by routinemodification of any of these methods.

5.4 Energy Transfer Dye Pairs

In another aspect, the present invention provides energy-transfer dyepairs incorporating the new rhodamine dyes of the invention. Generally,the energy-transfer dye pairs of the present invention comprise threemain elements: (i) a donor dye (DD) which absorbs light at a firstwavelength and emits excitation energy in response; (ii) an acceptor dye(AD) which is capable of absorbing the excitation energy emitted by thedonor dye and emitting light at a second wavelength in response; and(iii) a linkage linking the donor dye to the acceptor dye, the linkagebeing effective to facilitate efficient energy-transfer between thedonor and acceptor dyes. In the energy-transfer dye pairs of theinvention, at least one of the donor or acceptor dyes, typically theacceptor dye, is a new rhodamine dye of the invention. The donor andacceptor dyes can be linked together in a variety of differentconfigurations, depending upon the identities of the dyes. A thoroughdiscussion of the various structures, synthesis and use of certainenergy-transfer dye pairs which may aid an understanding of theenergy-transfer dye pairs of the invention is provided in U.S. Pat. No.5,800,996; U.S. Pat. No. 5,863,727; and U.S. Pat. No. 5,654,419, thedisclosures of which are incorporated herein by reference in theirentireties.

The energy-transfer dye pairs of the invention are typically obtained asillustrated in Scheme V:

In Scheme V, a dye 140 which includes an optional linker L″ and acomplementary functional group F_(x) is condensed with a rhodamine dyeof the invention according to structural formula (Ia) or (Ib) to yieldenergy-transfer dye pairs according to structural formulae (IIa) and(IIb), respectfully, where for example three pyridinium rings arepresent.

During the condensation, reactive group R_(x) and complementaryfunctional group F_(x) react to form covalent linkage R⁴¹. Thus, it willbe recognized by those of skill in the art that reactive group R_(x) andcomplementary functional group F_(x) can each constitute respectivemembers of the various pairs of complementary groups described inSection 5.3.1, such as various pairs of complementary electrophiles andnucleophiles. Preferably, one of R_(x) or F_(x) is an amine, thiol orhydroxyl group, most preferably an amine group, and the other one ofR_(x) or F_(x) is a group capable of reacting with an amine, thiol orhydroxyl, most preferably a carboxylic acid or a salt, ester oractivated ester thereof. Thus, a particularly preferred covalent linkageR⁴¹ is an amide of the formula —C(O)NR⁴⁵—, where R⁴⁵ is hydrogen or(C₁-C₆) alkyl.

In the energy-transfer dye pairs according to structural formulae (IIa)and (IIb), DD/AD represents either a donor dye or an acceptor dye.Whether DD/AD constitutes a donor or acceptor will depend on therespective excitation and emission properties of DD/AD and Y. Typically,DD/AD is a dye belonging to the xanthene (including fluorescein andrhodamine dyes), cyanine, phthalocyanine or squaraine classes of dyes.Alternatively, DD/AD can be a dye of the invention, preferably a dyeaccording to structural formula (Ia) or (Ib). The only requirement isthat dye DD/AD either be capable of absorbing excitation energy emittedfrom Y or be capable of emitting excitation energy absorbable by Y.Preferably, in addition to complementary functional group F_(x), DD/ADincludes a linking moiety or linking group as previously described forconjugating the energy-transfer dye pairs of the invention to othercompounds or substances. L″ represents a bond or a linker analogous tothe previously described linkers L that, when included in anenergy-transfer dye pair according to structural formula (IIa) or (IIb),facilitates efficient energy transfer between the acceptor and donorchromophores. The identity of L″ and its point of attachment to DD/ADwill depend, in part, upon the identity of DD/AD. Structures of thesevarious classes of AD/DD dyes, as well as suitable linkers L″ and pointsof attachment to these various classes of AD/DD dyes are described inU.S. Pat. No. 5,863,727, the disclosure of which is incorporated hereinby reference. When DD/AD is a cyanine dye, L″ is preferably an amide,and is attached to the quaternary nitrogen. When DD/AD is aphthalocyanine dye, L″ is preferably a sulfonamide, and is attached tothe aromatic carbon skeleton. When DD/AD is a squaraine dye, L″ ispreferably an amide, and is attached to the quaternary nitrogen. WhenDD/AD is a rhodamine dye, L″ is preferably an amide, and is attached toC4 or R⁹. When DD/AD is a fluorescein dye, U is preferably an amide, andis attached to C4 or R⁹.

Most preferably, DD/AD is a donor dye which emits excitation energyabsorbable by Y. Preferred donor dyes are xanthene dyes, especiallythose which include a linking moiety or linking group on their bottomrings. Preferred amongst the xanthene dyes are the fluorescein dyes.Fluorescein dyes suitable for use as donor dyes include, but are notlimited to, the 4,7-dichlorofluoresceins described in U.S. Pat. No.5,188,934, the extended fluoresceins described in U.S. Pat. No.5,775,409, 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM),5-(and 6)-carboxyfluorcscein (5,6-FAM), NAN, Cl-FLAN and TET. Linker L″is typically attached to the xanthene C4 position of these preferredfluorescein donor dye. The actual choice of donor dye will depend uponthe emission and excitation properties of DA/AD and Y, and will beapparent to those of skill in the art. In preferred fluorescein donordyes that include a 5-, 6- or 5-(and 6)-carboxyl, the carboxyl group (oran activated ester thereof) can be used to covalently conjugate theenergy-transfer dye pairs of the invention to other compounds orsubstances.

The energy-transfer dyes of the invention are more fully described belowwith reference to Various preferred embodiments. Referring to Scheme V,one group of preferred energy-transfer dye pairs according to structuralformulae (IIa) and (IIb) are obtained when the compounds according tostructural formulae (Ia) and (Ib) are any of their previously-describedpreferred embodiments and compound 140 has the structure:

-   -   including any counter ions, wherein:    -   R³³ is hydrogen a

-   -   R³⁴, when taken alone, is hydrogen, or when taken together with        R³⁵ is benzo;    -   R³⁵, when taken alone, is hydrogen, fluoro, chloro, hydroxyl or        carboxyl, or when taken together with R³⁴ is benzo;    -   R³⁶ is hydrogen, fluoro or chloro;    -   R³⁷, when taken alone, is hydrogen, fluoro, chloro, hydroxyl or        carboxyl, or when taken together with R³⁸ is benzo;    -   R³⁸, when taken alone, is hydrogen, or when taken together with        R³⁷ is Benzo;    -   R³⁹ is hydrogen or chloro;    -   R⁴⁰ is hydrogen or chloro; and    -   R⁵⁰ is carboxyl, or a salt, ester or activated ester thereof.        Preferred compound 140 can be the pure 5-isomer, the pure        6-isomer or a mixture of 5-(and 6)-isomers. Particularly        preferred compounds 140 are those in which R³⁴, R³⁵, R³⁶, R³⁷,        R³⁸, R³⁹ and R⁴⁰ are each hydrogen.

In another preferred embodiment, the energy-transfer dye pairs of theinvention are compounds according to structural formula (IIa) in which;

-   -   Y is a compound according to structural formula (Y-1), (Y-2),        (Y-3), (Y-4), (Y-20a), (Y-21a), (Y-22a), (Y-23a), (Y-24a),        (Y-25a), (Y-31a), (Y-34a), (Y-35a), (Y-36a), (Y-39a), (Y-41a),        (Y-42a), (Y-43a), (Y-44a), (Y-45a), or (Y-46a);    -   DD/AD is a fluorescein dye having a 5- or 6-carboxyl group or a        salt, ester or activated ester thereof, particularly a        fluorescein dye selected from the group consisting of        5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM),        5-(and -6)-carboxyfluorescein, NAN, Cl-FLAN and TET;    -   L is a bond, (C₁-C₆) alkyldiyl or (C₁-C₃) alkano, preferably a        bond;    -   Z¹ is (C₁-C₆)alkyldiyl, (C₅-C₁₄)aryl or heteroaryl;    -   R⁴¹ is —C(O)—R⁴², where R² is O, S or NH and is bonded to L″;        and/or    -   L″ is —R⁴¹—Z³—C(O)—R⁴⁴—R⁴⁵—, wherein R⁴³ is (C₁-C₆) alkyldiyl,        preferably (C₁-C₃) alkano, and is bonded to R⁴²; Z³ is 5-6        membered cyclic alkenyldiyl or heteroalkenyldiyl, (C₅-C₁₄)        aryldiyl or heteroaryldiyl; R⁴⁴ is O, S or NH; and R⁴⁵ is        (C₁-C₆) alkyldiyl, preferably (C₁-C₃) alkano, and is bonded to        the xanthene C4 carbon of DD/AD.

Particularly preferred amongst the above-described energy-transfer dyepairs are those compounds in which:

-   -   L is a bond;    -   Z¹ and Z³ are each independently selected from the group        consisting of phenyldiyl, phena-1,4-diyl, naphthyldiyl,        naphtha-2,6-diyl and naphtha-3,6-diyl;    -   R⁴² is NH;    -   R⁴³ is methano;    -   R⁴⁴ is NH; and/or    -   R⁴⁵ is methano.

In another preferred embodiment, the energy-transfer dye pairs of theinvention are compounds according to structural formula (IIb) in which:

-   -   E is carboxyl or a salt thereof,    -   Y is a compound according to structural formula (Y-1), (Y-2),        (Y-3) or (Y-4), where L is attached at position R^(3′), or is a        compound according to structural formula (Y-20b), (Y-21b),        (Y-22b), (Y-23b), (Y-24b), (Y-25b), (Y-31b), (Y-34b), (Y-35b),        (Y-36b), (Y-39b), (Y-41b), (Y-42b), (Y-43b), or (Y-46b);    -   DD/AD is a fluorescein dye having a 5- or 6-carboxyl group or a        salt, an ester or an activated ester thereof, particularly a        fluorescein dye selected from the group consisting of        5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM),        5-(and -6)-carboxyfluorescein, NAN, Cl-FLAN and TET;    -   Z is (C₁-C₆) alkyl, (C₁-C₆) alkanyl, (C₅-C₁₄) aryl or        heteroaryl;

L is or —CH₂-φ-, or —CH₂-Ψ-, where φ is phenyldiyl and Ψ is naphthyldiyland the methylene is attached to Y;

-   -   R⁴¹ is —C(O)—R⁴²—, where R⁴² is O, S or NH; and/or    -   L″ is —R⁴³—Z³—C(O)—R⁴⁴—R⁴³—, wherein R⁴³ is (C₁-C₆) alkyldiyl,        preferably (C₁-C₃) alkano, and is bonded to R⁴²; Z³ is 5-6        membered cyclic alkenyldiyl or heteroalkenyldiyl, (C₅-C₁₄)        aryldiyl or heteroaryldiyl; R⁴⁴ is O, S or NH; and R⁴⁵ is        (C₁-C₆) alkyldiyl, preferably (C₁-C₃) alkano, and is bonded to        the xanthene C4 carbon of DD/AD.

Particularly preferred amongst the above-described energy-transfer dyepairs are those compounds in which:

-   -   L is methano;    -   Z is selected from the group consisting of phenyl, naphthyl,        naphth-1-yl and naphth-2-yl;    -   Z³ is selected from the group consisting of phenyldiyl,        phen-1,4-diyl, naphthyldiyl, naphth-2,6-diyl and        naphth-3,6-diyl;    -   R⁴² is NH;    -   R⁴³ is methano;    -   R⁴⁴ is NH; and/or    -   R⁴⁵ is methano.

5.4.1 Synthesis of the Energy-Transfer Dye Pairs

Methods for synthesizing the various energy-transfer dyes of theinvention are illustrated in Scheme V, supra. Conditions for carryingout the reactions are described in U.S. Pat. No. 5,863,727, includingCompound 140. Syntheses of exemplary energy-transfer dye pairs aredescribed in the Examples section. All of the energy-transfer dye pairsof the invention can be obtained by routine modification of any of thesemethods.

5.5 Conjugates Incorporating Dyes and Energy-Transfer Dye Pairs

In another aspect, the present invention comprises molecules and/orsubstances or conjugated with the rhodamine dyes and/or energy-transferdye pairs of the invention. The conjugates can comprise virtually anymolecule or substance to which the dyes or energy-transfer dye pairs ofthe invention can be conjugated, including by way of example and notlimitation, peptides, polypeptides, proteins, saccharides,polysaccharides, nucleosides, nucleotides, polynucleotides, lipids,solid synthesis supports, organic and inorganic polymers, andcombinations and assemblages thereof, such as chromosomes, nuclei,living cells (e.g., bacteria or other microorganisms, mammalian cells,tissues, etc.), and the like. The dyes or energy-transfer dye pairs areconjugated with the reagent via a linking moiety by a variety of means,including hydrophobic attraction, ionic attraction, covalent attachmentor with the aid of pairs of specific binding molecules, as previouslydescribed. Preferably, the dyes are conjugated via covalent attachment.

Conjugation typically results from mixing a dye or dye pair including anoptional linking moiety with and the molecule or substance to beconjugated in a suitable solvent in which both are soluble, usingmethods well-known in the art, followed by separation of the conjugatefrom any unconjugated starting materials or unwanted by-products. Thedye or dye pair conjugate can be stored dry or in solution for lateruse.

5.5.1 Nucleoside/Tide Conjugates

A preferred class of conjugates include nucleosides/tides andnucleoside/tide analogs that are labeled with the rhodamine dyes orenergy-transfer dye pairs of the invention. Such labelednucleosides/tides are particularly useful for labeling polynucleotidesformed by enzymatic synthesis, e.g., labeled nucleotide triphosphatesused in the context of template-directed primer extension, PCRamplification, Sanger-type polynucleotide sequencing, and/ornick-translation reactions. Labeled nucleoside/tides and/ornucleoside/tide analog conjugates are generally obtained by condensing anucleoside/tide or nucleoside/tide analog (NUC) modified to contain alinking moiety (-L′-F_(x)) with a rhodamine dye according to structuralformula (Ia) or (Ib) to yield a dye-labeled nucleoside/tide ornucleoside/tide analog according to structural formula (IIIa) or (IIIb),respectively. Energy-transfer dye pair-labeled nucleosides/tides and/ornucleoside/tide analogs are obtained in an similar manner withenergy-transfer dye compounds according to structural formulae (IIa) and(IIb) in which the DD/AD dye includes an optional linking moiety of theformula -L³-R_(x), where is a bond or linker similar topreviously-described linker L (compounds (IIa.1) and (IIb.1),respectively). These reactions are illustrated in Schemes VIa and Vib:

Referring to Schemes VIa and VIb, where for example three pyridiniumrings are present and reactive functional group R_(x) on dyes orenergy-transfer dye pairs according to structural formulae (Ia), (Ib),(IIa.1) and/or (IIb.1) and complementary functional group F_(x) oncompound 144 react to form covalent linkage R⁴⁶. Covalent linkage R⁴⁶ isanalogous to the previously-described covalent linkage R⁴¹. Preferredembodiments of R⁴⁶ are the same as the preferred embodiments previouslydescribed for R⁴¹.

Complementary functional group F_(x) is attached to NUC via linker L′.Complementary functional group F_(x) may be attached directly to NUC, inwhich case L′ represents a bond, or it may be spaced away from NUC byone or more intervening atoms, in which case L′ represents a linker. Anyof the linkers L or L″ previously described in connection with therhodamine dye compounds per se and/or the energy-transfer dye pairs perse can be used for linker L′.

Complementary functional group F_(x) may be attached to NUC at a varietyof different positions, e.g., the nucleobase, the sugar and/or thephosphate ester moiety. Nucleosides/tides and nucleoside/tide analogsthat are appropriately modified at these various positions such thatthey can be conjugated with dyes and/or dye pairs according to theinvention are known in the art. Preferably, complementary group F, isattached to the nucleobase. When the nucleobase is a 7-deazapurine, L′is usually attached to the C7 position of the nucleobase. When thenucleobase is a pyrimidine, L′ is usually attached to the C-5 positionof the nucleobase. When the nucleobase is a purine, L′ is usuallyattached to the C-8 position of the nucleobase. Linkers L′ useful forcovalently conjugating the dyes of the invention to the nucleobase ofNUC are described in U.S. Pat. No. 5,821,356, U.S. Pat. No. 5,770,716and U.S. application Ser. No. 08/833,854 filed Apr. 10, 1997, thedisclosures of which are incorporated herein by reference.

Preferred linkers L′ for covalently conjugating the dyes of theinvention to the nucleobase of NUC include (C₂-C₂₀)alkylenos,heteroalkyldiyls and heteroalkylenos, especially (C₂-C₂₀) alkynos,(C₂-C₂₀) alkenos, heteroalkynos and heteroalkenos. A particularlypreferred linker L′ is —C≡C—CH₂—, where the terminal sp carbon iscovalently attached to the nucleobase of NUC and the terminal methylene(sp³) carbon is covalently attached to R⁴⁶ in the compounds ofstructural formulae (IIIa), (IIIb), (IVa) and (IVb), or to F_(x) ofcompound 144.

Additional preferred linkers L′ for covalently conjugating the rhodaminedyes or energy-transfer dye pairs of the invention to the nucleobase ofNUC include propargylethoxy groups according to structural formula—C≡C—CH₂—O—CH₂—CH₂—NR⁴⁷—R⁴⁸—, wherein R⁴⁷ is hydrogen or (C₁-C₆) alkyland R⁴⁸ is selected from the group consisting of —C(O)—(CH₂)_(r)—,—C(O)—CHR⁴⁹—, —C(O)—C≡C—CH₂— and —C(O)-φ-(CH₂)_(r)—, where each r isindependently an integer from 1 to 5 and φ represents a C₆ aryldiyl orheteroaryldiyl, preferably phena-1,4-diyl

and R⁴⁹ is hydrogen, (C₁-C₆) alkyl or an amino acid side chain(including side chains of both gene-encoded and non-encoded aminoacids). With these linkers L′, the terminal sp carbon is attached to thenucleobase of NUC and the other terminal group is attached to R⁴⁶ in thecompounds of structural formulae (IIIa), (IIIb), (IVa) and (IVb), or toF_(x) of compound 144.

In a preferred embodiment, the labeled nucleosides/tide and/ornucleoside/tide analogs according to structural formulae (IIIa), (IIIb),(IVa) and (IVb) are labeled enzymatically-incorporatable nucleotides,labeled enzymatically extendable nucleotides or labeled terminators.

In another preferred embodiment, the labeled nucleosides/tides andnucleoside/tide analogs are those obtained from Schemes (VIa) and (VIb)in which the compounds according to structural formulae (Ia), (Ib) areany of their respective previously-defined preferred embodiments,compounds (IIa.1) and (IIb.1) are any of the previously definedpreferred embodiments of compounds (IIa) and (IIb), respectively, andcompound 144 is a compound according to structural formula (V):

wherein:

-   -   B is a nucleobase;    -   F_(x) is a complementary functional group as previously        described;    -   L′ is one of the preferred linkers described above;    -   R₇₀ and R₇₁, when taken alone, are each independently selected        from the group consisting of hydrogen, hydroxyl and a moiety        which blocks polymerase-mediated template-directed nucleic acid        synthesis, or when taken together form a bond such that the        illustrated sugar is 2′,3′-didehydroribose; and    -   R₇₂ is selected from the group consisting of hydroxyl, a        phosphate ester having the formula

where a is an integer from 0 to 2, and a phosphate ester analog.Typically, F_(x) is an amino group of the formula —NHR⁵¹, where R⁵¹ ishydrogen or (C₁-C₆)alkyl, but can be any nucleophilic or electrophilicgroups.

In a preferred embodiment of structural formula (V), B is a normalnucleobase or a common analog thereof, a 7-deazapurine, 8-aza,7-deazapurine, a purine or a pyrimidine. In a particularly preferredembodiment, B is a nucleobase selected from the group consisting of7-deaza-adenine, cytosine, 7-deaza-guanine, thymine and uracil. When thepreferred nucleobase B is a purine or a 7-deaza-purine, the pentosemoiety is attached to the N⁹-position of the nucleobase, and when thepreferred B is a pyrimidine, the pentose moiety is attached to theN¹-position of the nucleobase. Linker L′ is attached to nucleobase B aspreviously described.

In structural formula (V), when both R₇₀ and R₇₁ are hydroxyl, theresultant compounds produced in Schemes VIa and VIb are labeledribonucleoside/tides. When R₇₀ is hydrogen and R₇₁ is hydroxyl, theresultant compounds are labeled 2′-deoxyribonucleoside/tides. When R₇₀and R₇₁ are each hydrogen, the resultant compounds are2′,3′-dideoxyribonucleoside/tides. Labeled2′,3′-dideoxyribonucleoside-5′-triphosphates (ddNTPs) find particularuse as terminators in Sanger-type DNA sequencing methods utilizingfluorescent detection. Labeled 2′-deoxyribonucleoside-5′-triphosphates(dNTPs) find particular use as means for labeling DNA polymeraseextension products, e.g., in the polymerase chain reaction ornick-translation. Labeled ribonucleoside-5′-triphosphates (NTPs) findparticular use as means for labeling RNA polymerase extension products.

Referring to Schemes (VIa) and (VIb), supra, the synthesis ofalkynylamino-derivatized compounds 144 useful for conjugating the dyesof the invention to nucleosides/tides and/or nucleoside/tide analogs istaught in EP 87305844.0 and Hobbs et al., 1989, J. Org. Chem. 54:3420.The corresponding nucleoside mono-, di- and triphosphates are obtainedby standard techniques (see, e.g., the methods described in U.S. Pat.No. 5,821,356, U.S. Pat. No. 5,770,716 and U.S. application Ser. No.08/833,854 filed Apr. 10, 1997, discussed supra). Methods forsynthesizing compound 144 in modified with propargylethoxyamido linkersL′ can also be found in these patents and application.

Energy-transfer dye pairs can be conjugated to a nucleotide5′-triphosphate 144 by linking through a nucleobase amino group to: (i)an activated ester of a energy-transfer dye pair, e.g. 230 NHS ester, or(ii) stepwise coupling to one dye 140, e.g. 4′-protected aminomethylfluorescein, then coupling the unprotected 4′-aminomethyl to the seconddye of the pair, e.g. 196 NHS ester.

Additional synthesis procedures suitable for use in synthesizingcompounds according to structural formulae (IIIa), (IIIb), (IVa) and(IVb) are described, for example, in Gibson et al., 1987, Nucl. AcidsRes. 15:6455-6467; Gebeyehu et al., 1987, Nucl. Acids Res. 15:4513-4535;Haralambidis et al., 1987, Nucl. Acids Res. 15:4856-4876; Nelson et al.,1986, Nucleosides and Nucleotides. 5(3):233-241; Bergstrom et al., 1989,J. Am. Chem. Soc. 111:374-375; U.S. Pat. No. 4,855,225, U.S. Pat. No.5,231,191 and U.S. Pat. No. 5,449,767, the disclosures of which areincorporated herein by reference. Any of these methods can be routinelyadapted or modified as necessary to synthesize the full range of labelednucleosides/tides and nucleoside/tide analogs described herein.

5.5.1.1 Poly-nucleotide Conjugates

Yet another preferred class of conjugates of the present inventioncomprise polynucleotides and/or polynucleotide analogs labeled with therhodamine dyes or energy-transfer dye pairs of the invention. Suchlabeled polynucleotides and/or analogs are useful in a number ofimportant contexts, including as DNA sequencing primers, PCR primers,oligonucleotide hybridization probes, oligonucleotide ligation probes,and the like.

In one preferred embodiment, the labeled polynucleotides orpolynucleotide analogs of the present invention include multiple dyeslocated such that fluorescence energy-transfer takes place between adonor dye and an acceptor dye. Such multi-dye-energy-transferpolynucleotides find application as spectrally-tunable sequencingprimers as described, for example, in Ju et al., 1995, Proc. Natl. Acad.Sci. USA 92:4347-4351, and as hybridization probes as described, forexample, in Lee et al., 1993, Nucl. Acids Res. 21:3761-3766.

Labeled polynucleotides and/or polynucleotide analogs are typicallysynthesized enzymatically, e.g., using a DNA/RNA polymerase or ligase(see, e.g., Stryer, 1981, Biochemisty, Chapter 24, W.H. Freeman andCompany) and a labeled enzymatically-incorporatable nucleotide, aspreviously described. Alternatively, the labels may be introducedsubsequent to synthesis via conventional conjugation reactions, asdiscussed more thoroughly below.

Generally, if the labeled polynucleotide is made using enzymaticsynthesis, the following procedure may be used. A target DNApolynucleotide is denatured and an oligonucleotide primer is annealed tothe target DNA. A mixture of 2′-deoxyribonucleoside-5′-triphosphatescapable of supporting template-directed enzymatic extension of theprimed target (e.g., a mixture including dGTP, dATP, dCTP, and dTTP) isadded to the primed target. At least a fraction of the deoxynucleotidesare labeled with a rhodamine dye or energy-transfer dye pair of theinvention as described above. Next, a polymerase enzyme is added to themixture under conditions where the polymerase enzyme is active. Alabeled polynucleotide is formed by the incorporation of the labeleddeoxynucleotides during polymerase-mediated strand synthesis. In analternative enzymatic synthesis method, two primers are used instead ofone: one complementary to the plus (+) strand of the target and anothercomplementary to the minus (−) strand of the target, the polymerase is athermostable polymerase and the reaction temperature is cycled between adenaturation temperature and an extension temperature, therebyexponentially synthesizing a labeled complement to the target sequenceby PCR (see, e.g., PCR Protocols, 1990, Innis et al. Eds., AcademicPress).

Alternatively, the labeled polynucleotide or polynucleotide analog isobtained via post-synthesis conjugation. According to this embodiment, apolynucleotide or polynucleotide analog which includes a complementaryfunctional group F_(x), typically an amino group, is condensed with arhodamine dye according to structural formula (IIa) or (IIb) or anenergy-transfer dye pair according to structural formula (IIIa) or(IIIb) under conditions wherein R_(x) and F_(x) react to form a covalentlinkage. The labeled polynucleotides and/or polynucleotide analogs areisolated using conventional means, such as alcohol precipitation, gelelectrophoresis, column chromatography, etc.

A variety of reagents for introducing amino groups, or othercomplementary functional groups such as thiols into enzymatically orchemically synthesized polynucleotides and polynucleotide analogs areknown in the art, as are appropriate condensation conditions. Forexample, methods for labeling polynucleotides at their 5′-terminus aredescribed in Oligonucleotides and Analogs, 1991. Eckstein, Ed., Chapter8, IRL Press; Orgel et al., 1983, Nucl. Acids Res. 11(18):6513; and U.S.Pat. No. 5,118,800. Methods for labeling polynucleotides at thephosphate ester backbone are described in Oligonucleotides and Analogs,1991. Eckstein, Ed., Chapter 9, IRL Press. Methods for labelingpolynucleotides at their 3′-terminus are described in Nelson et al.,1992, Nucl. Acids Res. 20(23):6253-6259; U.S. Pat. No. 5,401,837; andU.S. Pat. No. 5,141,813. For a review of labeling procedures, the readeris referred to Haugland, In: Excited States of Biopolymers, 1983,Steiner. Ed., Plenum Press, NY. All of these disclosures areincorporated herein by reference.

5.6 Methods Utilizing the Dyes and Reagents of the Invention

The rhodamine dyes, energy-transfer dye pairs and conjugatesincorporating the dyes and energy-transfer dye pairs of the presentinvention are well suited to any method utilizing fluorescent detection,particularly aqueous applications and methods requiring the simultaneousdetection of multiple spatially-overlapping analytes. The various dyesand conjugates of the invention are particularly well suited foridentifying classes of polynucleotides that have been subjected to abiochemical separation procedure, such as electrophoresis, or that havebeen distributed among locations in a spatially-addressablehybridization array.

The various dyes of the invention can be conjugated to peptides,proteins, antibodies, and antigens. Dye-antibody conjugates are awfulfor sandwich-type immunosorbent assays. Especially preferred arebead-based assays for detection of peptides, cells, and other cellularcomponents where the dyes of the invention are conjugated to antibodies.

In a preferred category of methods referred to herein as “fragmentanalysis” or “genetic analysis” methods, labeled polynucleotidefragments are generated through template-directed enzymatic synthesisusing labeled primers or nucleotides, e.g., by ligation orpolymerase-directed primer extension; the fragments are subjected to asize-dependent separation process, e.g., electrophoresis orchromatography, and, the separated fragments are detected subsequent tothe separation, e.g., by laser-induced fluorescence. In a particularlypreferred embodiment, multiple classes of polynucleotides are separatedsimultaneously and the different classes are distinguished by spectrallyresolvable labels.

One such fragment analysis method known as amplified fragment lengthpolymorphism detection (AmpFLP) is based on amplified fragment lengthpolymorphisms, i.e., restriction fragment length polymorphisms that areamplified by PCR. These amplified fragments of varying size serve aslinked markers for following mutant genes through families. The closerthe amplified fragment is to the mutant gene on the chromosome, thehigher the linkage correlation. Because genes for many genetic disordershave not been identified, these linkage markers serve to help evaluatedisease risk or paternity. In the AmpFLPs technique, the polynucleotidesmay be labeled by using a labeled polynucleotide PCR primer, or byutilizing labeled nucleotide triphosphates in the PCR.

In another such fragment analysis method known as nick translation, areaction is used to replace unlabeled nucleotides in a double-stranded(ds) DNA molecule with labeled nucleotides. Free 3′-hydroxyl groups arecreated within the dsDNA by “nicks” caused by treatment withdeoxyribonuclease I (DNAase I). DNA polymerase I then catalyzes theaddition of a labeled nucleotide to the 3′-hydroxyl terminus of thenick. At the same time, the 5′ to 3′-exonuclease activity of this enzymeeliminates the nucleotide at the 5′-phosphoryl terminus of the nick. Anew nucleotide with a free 3′-OH group is incorporated at the positionof the original excised nucleotide, and the nick is shifted along by onenucleotide in the 3′ direction. This 3′ shift will result in thesequential addition of new labeled nucleotides into the dsDNA. Thenick-translated polynucleotide is then analyzed, for example, by using aseparation process such as electrophoresis.

Another exemplary fragment analysis method is based on variable numbersof tandem repeats, or VNTRs. VNTRs are regions of double-stranded DNAthat contain multiple adjacent copies of a particular sequence, with thenumber of repeating units being variable. Examples of VNTR loci arepYNZ22, pMCT118, and Apo B. A subset of VNTR methods are those methodsbased on the detection of microsatellite repeats, or short tandemrepeats (STRs), i.e.; tandem repeats of DNA characterized by a short(2-4 bases) repeated sequence. One of the most abundant interspersedrepetitive DNA families in humans is the (dC-dA)n-(dG-dT)n dinucleotiderepeat family (also called the (CA)n dinucleotide repeat family). Thereare thought to be as many as 50,000 to 100,000 (CA)n repeat regions inthe human genome, typically with 15-30 repeats per block. Many of theserepeat regions are polymorphic in length and can therefore serve asuseful genetic markers. Preferably, in VNTR or STR methods, label isintroduced into the polynucleotide fragments using a labeled PCR primer.

In a particularly preferred fragment analysis method, classes identifiedin accordance with the invention are defined in terms of terminalnucleotides so that a correspondence is established between the fourpossible terminal bases and the members of a set of spectrallyresolvable dyes. Such sets are readily assembled from the dyes andenergy-transfer dye pairs of the invention by measuring emission andabsorption bandwidths with commercially available spectrophotometers.More preferably, the classes arise in the context of the chemical orchain termination methods of DNA sequencing, and most preferably theclasses arise in the context of the chain termination methods, i.e.,dideoxy DNA sequencing, or Sanger-type sequencing.

Sanger-type sequencing involves the synthesis of a DNA strand by a DNApolymerase in vitro using a single-stranded or double-stranded DNAtemplate whose sequence is to be determined. Synthesis is initiated at adefined site based on where an oligonucleotide primer anneals to thetemplate. The synthesis reaction is terminated by incorporation of aterminator that will not support continued DNA elongation. When properproportions of dNTPs and a single terminator complementary to A, G, C orT are used, enzyme-catalyzed primer extension will be terminated in afraction of the extension products at each site where the terminator isincorporated. If labeled primers or labeled terminators are used foreach reaction, the sequence information can be detected by fluorescenceafter separation of the resultant primer extension products byhigh-resolution electrophoresis. In the chain termination method, dyesor energy-transfer dye pairs of the invention can be attached to eitherthe sequencing primers or terminators. The dyes or energy-transfer dyepairs can be linked to a complementary functionality on the 5′-end ofthe primer, e.g. following the teaching in Fung et al, U.S. Pat. No.4,757,141; on the base of a primer; or on the base of a terminator, e.g.via the alkynylamino or other linking groups described above.Concentration ranges for the various enzymes, primers, dNTPs and labeledterminators are those commonly employed in the art.

In each of the above fragment analysis methods, labeled extensionproducts are preferably separated by electrophoretic procedures, e.g.Gel Electrophoresis of Nucleic Acids: A Practical Approach, 1981,Rickwood and Hames, Eds., IRL Press Limited, London; Osterman, 1984,Methods of Protein and Nucleic Acid Research, Vol. 1 Springer-Verlag,Berlin; or U.S. Pat. Nos. 5,374,527, 5,624,800 and/or 5,552,028.Preferably, the type of electrophoretic matrix is crosslinked oruncrosslinked polyacrylamide having a concentration (weight to volume)of between about 2-20 weight percent. More preferably, thepolyacrylamide concentration is between about 4-8 percent. Preferably,in the context of DNA sequencing, the electrophoresis matrix includes adenaturing agent, e.g., urea, formamide, and the like. Detailedprocedures for constructing such matrices are given by Maniatis at al.,1980, “Fractionation of Low Molecular Weight DNA and RNA inPolyacrylamide Gels Containing 98% Formamide or 7 M Urea,” Methods inEnzymology 65:299-305; Maniatis et al., 1975, “Chain LengthDetermination of Small Double- and Single-Stranded DNA Molecules byPolyacrylamide Gel Electrophoresis,” Biochemistry 14:3787-3794; Maniatiset al., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York, pgs. 179-185; and ABI PRISM™ 377 DNA SequencerUser's Manual, Rev. A, January 1995, Chapter 2 (p/n 903433, ThePerkin-Elmer Corporation, Foster City, Calif.). The optimalelectrophoresis conditions, e.g., polymer concentration, pH,temperature, and concentration of denaturing agent, employed in aparticular separation depend on many factors, including among others,the size range of the nucleic acids to be separated, their basecompositions, whether they are single stranded or double stranded, andthe nature of the classes for which information is sought byelectrophoresis. Accordingly, application of the invention may requirestandard preliminary testing to optimize conditions for particularseparations.

Subsequent to electrophoretic separation, the labeled extension productsare detected by measuring the fluorescence emission from the labels. Toperform such detection, the labeled products are illuminated by standardmeans, e.g. high intensity mercury vapor lamps, lasers, or the like. Theillumination wavelength will depend upon the spectral properties of theparticular label, Preferably, the illumination means is a laser havingan illumination beam at a wavelength between 400 and 700 nm. Morepreferably, the illumination means is a laser light generated by anargon ion laser, particularly the 488 and 514 nm emission lines of anargon ion laser, or the 532 emission line of a neodymium solid-state YAGlaser or the 633 nm emission line of a helium-neon laser. Several argonion lasers are available commercially which lase simultaneously at theselines, e.g. Cyonics, Ltd. (Sunnyvale, Calif.) Model 2001, or the like.The fluorescence is then detected by a light-sensitive detector, e.g., aphotomultiplier tube, a charged coupled device, or the like. Suitableexemplary electrophoresis detection systems are described elsewhere,e.g., U.S. Pat. Nos. 5,543,026; 5,274,240; 4,879,012; 5,091,652 and4,811,218.

In preferred embodiments, the primer is unlabeled and the sequencingreaction includes, in addition to the polymerase and mixture of dNTPs, amixture of four different terminators, one complementary to A, onecomplementary to G, one complementary to C and one complementary to T.Each of the different terminators is labeled with a different,spectrally resolvable dye or energy-transfer dye pair. One of theterminators is labeled with a rhodamine dye or energy-transfer dye pairof the invention. As each of the labeled terminators fluoresces at adifferent wavelength, following separation based on size, the identityof the 3′-terminal nucleotide of each extension product is identified bythe wavelength (or color) of the label. In particularly preferredembodiments, each of the different spectrally resolvable labels can beexcited using a single light source. A set of such preferred labeledterminators is provided in the Examples section. Other sets will dependupon the excitation and emission spectral properties of the variouslabels are readily obtained by routine methods.

The invention having been described, the following examples are provideto illustrate, and not limit, the invention.

6. Example Synthesis of Rhodamine Dye 190

Rhodamine dye 190 was synthesized as illustrated in Schemes I and II,supra, from the appropriate aminophenol starting materials.

6.1 Synthesis of 202

Amino-phenol compound 200 was synthesized as described in U.S. Pat. No.5,750,409 by alkylation of 192 with ethyl iodide and sodium bicarbonatein acetonitrile followed by demethylation with boron tribromide indichloromethane.

A solution of aminophenol 200 (4.8 g, 22 mmol) and tetrafluorophthalicanhydride 114 (4.9 g, 22 mmol; Aldrich) was refluxed in toluene (44 ml)for 3 hr. The solution was cooled to rt and the precipitate collected toyield tetrafluoro ketone 202 (6.8 g, 70%).

6.2 Synthesis of 204

A suspension of the tert-butyl ester amine 194, prepared according toU.S. Pat. No. 5,688,808, (5.46 g, 20 mmol), methyl4-(bromomethyl)benzoate (8.3 g, 36.2 mmol), sodium iodide (3.0 g, 20mmol) and potassium carbonate (2.8 g, 20 mmol) was refluxed inacetonitrile (20 ml) for 1 hr. TLC (hexane/ethyl acetate 1/1) showed thereaction was complete. Ether was added to the cooled mixture and thesolution was decanted from the solids. The solution was washed withwater (2 portions of 50 ml), brine (50 ml) and dried over magnesiumsulfate. The crude product was purified by chromatography on silica gel(hexane/ethyl acetate 19:1) to give white crystals of the alkylated,tert-butyl ester product (4.3 g, 10 mmol, 50%). The tert-butyl ester wasdissolved in a solution of lithium hydroxide monohydrate (1.7 g, 40mmol) in water (10 ml) and methanol (50 ml), refluxed for 2 hr and thenevaporated to dryness under vacuum. The residue was extracted withdiethylether (100 ml) and washed with water and brine. The organic layerwas dried over magnesium sulfate, filtered and evaporated to give 204 asa tan foam. ¹H NMR (204, CD₂Cl₂) δ 8.03, 2H, d; 7.45, 2H, d; 6.96, 1H,d; 6.04, 1H, d; 5.65, 1H, s; 4.55, 1H, m; 1.38, 3H, s; 1.25, 6H, s.

6.3 Synthesis of 206

Phosphorous oxytrichloride (2.8 ml, 30 mmol) was added to a suspensionof tetrafluoro ketone 202 (4.4 g, 10 mmol) in chloroform (100 ml). Thesuspension was stirred at rt for 30 min., aminophenol 204 (3.2 g, 10mmol) was added and the mixture was refluxed for 3 hr. The solution wascooled to rt and the reaction quenched with water (1 ml). The solventwas evaporated and dye 206 was purified by normal phase chromatography(DCM/MeOH/HOAc, 90:10:1). Dye 206 was further purified by C18 reversephase chromatography (MeOH/0.1 M TEAA, 4:1) to afford a dark green solid(0.65 g, 9%, Abs. max 610 nm, Em. max 632 nm, H₂O).

6.4 Synthesis of Dye 190

Dimethylaminopyridine (85 mg) was added to tetrafluoro dye 206 (30 mg)in 0.7 ml of dimethylformamide. After 20 hours at room temperature, TLCanalysis (CH₂Cl₂:CH₃OH:CH₃CO₂H 80:20:16) showed the completedisappearance of starting 206 and the appearance of a new, lower Rfspot, the tetradimethylaminopyridinium adduct. Thiophenol (0.75 ml) wasadded, and after another 8 hours, TLC showed partial conversion to ahigher Rf spot. After concentration under vacuum, 190 was purified byreverse-phase HPLC (C18) by a gradient of 30-50% acetonitrile in 0.1MTEAA. The fractions containing 190 were combined and evaporated to givea blue oil which was precipitated in either to provide pure dye 190 as ablue solid (30 mg, Abs. Max 628 nm, Em. max 650 nm, H₂O).

The succinimidyl (NHS) ester of 190 was prepared from a solution of 190(5 mg) in 100 μl dimethylformamide (DMF). Succinimidyltetramethyluronium tetrafluoroborate (20 mg) and diisopropylamine (10μl) were added. After 1 hour at room temperature, TLC analysis(C2-reverse phase plates, CH₃OH/0.1M TEAA 1:1) showed the disappearanceof starting 190 and a new, lower Rf spot. The NHS ester of 190 wasisolated by precipitation as a blue solid.

7. Example Synthesis of Rhodamine Dye 196

Rhodamine dye 196 was synthesized in a manner analogous to dye 190 fromthe corresponding tetrafluoro dye 220.

7.1 Synthesis of Tetrafluoro 220

Phosphorous oxytrichloride (2.8 ml, 30 mmol) was added to a suspensionof tetrafluoro ketone 202 (4.4 g, 10 mmol) in chloroform (100 ml). Thesuspension was stirred at rt for 30 min., aminophenol 200 (3.2 g, 10mmol) was added and the mixture was refluxed for 3 hr. The solution wascooled to rt and the reaction quenched with water (1 ml). The solventwas evaporated and dye 220 was purified by normal phase chromatography(DCM/MeOH/HOAc, 90:10:1). Dye 220 was further purified by C18 reversephase chromatography (CH₃OH: 0.1M TEAA, 4:1) to afford a dark greensolid (0.65 g, 9%, Abs. max 613 nm, Em. max 643 nm, H₂O).

7.2 Synthesis of Rhodamine Dye 196

To a solution of 220 (100 mg) in dimethylformamide (1.5 mL) was added4-(dimethylamino)pyridine (150 mg). Thin-layer chromatography (TLC) onsilica gel eluting with 80:20:16 dichloromethane:methanol:acetic acidcould distinguish tetrafluoro 220 (Rf=1) from thetetra-dimethylaminopyridinium adduct (Rf=0). After 40 hr, analysis bythin-layer chromatography showed complete conversion to the SymJAZadduct.

To this solution, 4-carboxythiobenzene (50 mg) was added. After 10 min,TLC analysis showed conversion to rhodamine dye 196 (Rf=0.1).Purification of 196 was accomplished on C18 silica gel with stepwiseelution with 20-70% methanol vs. 0.1 M triethylammonium acetate. The dye196 eluted between 30% and 50% methanol. The solvent was evaporated andthe residual blue oil was precipitated with ether to provide the dye 196as a blue solid (85 mg, Abs. max 636 nm, Em. max 661 nm, H₂O).

7.3 Synthesis of Tetrafluoro 198

Amino-phenol compound 228 was synthesized as described in U.S. Pat. No.5,750,409 and cyclized with tetrafluorophthalic anhydride 114 as inExample 6.1 supra to give 238 (lambda max 384 nm). The mixture of 2.6mmole 238 and 2.6 mmole 228 in 1.2 gm phosphorus oxytrichloride and 10ml acetonitrile was refluxed for 12 hours. The solvent was evaporatedunder vacuum, dissolved in several milliliters of dichloromethane andchromatographed on silica gel, eluting with dichloromethane andmethanol. The product fractions were combined, evaporated and furtherpurified by reverse-phase preparative HPLC and precipitation in 1% HClto give 35 mg 198 as a red solid. ¹H NMR (198, CD₃OD) δ 6.82, 2H, s;6.59, 2H, s; 5.63, 2H, s; 1.9, 6H, br s; 1.4, 12H, br s.

7.4 Synthesis of Tetrafluoro 290

Tetrafluoro 240 was synthesized by the method and correspondingmethylated intermediates of Example 7.3. Mass spectroscopy 240: ExactMass=662.22 (Molecular Wt.=662.67) Ex. max 624 nm, Em. max 644 nm (8Murea).

8. Example Synthesis of Rhodamine Dye 232

Rhodamine dye 232 was synthesized as illustrated in Schemes (I) and(II), supra, from the appropriate aminophenol starting materials.

8.1 Synthesis of Tricyclic Amine-Phenol 208

A suspension of the tert-butyl ester amine 194, prepared according toU.S. Pat. No. 5,688,808, (12.8 g, 47 mmol), 1-bromo-3-chloropropane(29.3 g, 187 mmol), sodium iodide (56.4 g, 376 mmol) and sodiumbicarbonate (7.9 g, 94 mmol) was refluxed in acetonitrile (150 ml) for18 hr. The mixture was cooled to room temperature, filtered by suctionfiltration and evaporated to leave a residue. The filter cake was washedwith hexane (300 ml) and the filtrate was combined with the residue andwashed with water (2 portions of 50 ml), brine (50 ml) and dried overmagnesium sulfate. The crude cyclized product was purified bychromatography on silica gel (hexane/ethyl acetate 20:1) to leave theintermediate tricyclic pivalate ester as a pale yellow oil (9.5 g, 30mmol, 64%). The cyclized ester was dissolved in a solution of lithiumhydroxide monohydrate (2.6 g, 60 mmol) in water (15 ml) and methanol(120 ml). The solution was stirred at room temperature for 1 hr and thenevaporated to dryness under vacuum. The residue was treated with 1 M HCl(30 ml) and extracted with diethylether (3 portions of 100 ml). Thecombined extracts were washed with 200 mM pH 7 phosphate buffer (50 ml),dried over magnesium sulfate, filtered and evaporated to give tricyclicamine-phenol 208 as a brown solid, used crude in subsequent steps.

8.2 Synthesis of Tetrafluoro Ketone 210

A solution of aminophenol 208 (0.67 g, 2.9 mmol) and tetrafluorophthalicanhydride (0.64 g, 2.9 mmol) was refluxed in toluene (10 ml) for 3 hr.The solution was cooled to rt and the precipitate collected, yieldingtetrafluoro ketone 210 (0.92 g, 71%).

8.3 Synthesis of Tetrafluoro 212

Phosphorous oxytrichloride (0.56 ml, 6 mmol) was added to a solution ofF4 RAZ ketone 210 (0.91 g, 2 mmol) in chloroform (20 ml). The solutionwas stirred for 15 min, aminophenol 208 (0.46 g, 2 mmol) was added andthe mixture was refluxed for 3 hr. The solution was cooled to rt and thereaction quenched with water (0.5 ml). Tetrafluoro dye 212 was purifiedby normal phase chromatography (DCM/MeOH, 20:1) and further purified byC18 reverse phase chromatography (MeOH/0.1 M TEAA, 9:1) to afford 212 asa metallic green solid (0.39 g, 30%, Abs. max 630 nm, Em. max 655 nm, 8Murea).

8.4 Synthesis of Rhodamine Dye 232

Rhodamine dye 232 was synthesized from tetrafluoro dye 212 with4-(dimethylamino)pyridine and 4-carboxythiobenzene, as described inSection 7.2, supra, and purified by C-8 reverse phase HPLC, eluting withan acetonitrile/0.1 M TEAA gradient (Em. max 665 nm, H₂O).

9. Example Synthesis of Rhodamine Dye 234

Rhodamine dye 234 was synthesized as illustrated in Schemes (I) and (II)from the appropriate aminophenol starting materials.

9.1 Synthesis of Pyrrolidinyl Phenol 214

A solution of m-aminophenol (12.6 g, 115 mmol) was heated in1,4-dibromobutane (50 g, 230 mmol) at 130° C. for 18 hr. The mixtureswas cooled to room temperature and the gum was triturated with ether andthen ethyl acetate. The gum was dissolved in 120 ml of 1 M NaOH andextracted with ethyl acetate (100 ml). The layers were separated and theorganic phase was washed with water twice and then brine. After dryingover magnesium sulfate, the crude product was purified by silica gelchromatography (DCM/methanol 100:1) to give a pale yellow solid. Thesolid was refluxed in toluene (500 ml) and triethylamine (16 ml, 115mmol) for 2 hr., cooled to room temperature, and washed with water. Thesolvent was evaporated to leave pyrrolidinyl phenol 214 as a white solid(11 g, 60%). ¹H NMR (214, d6-DMSO) δ 8.98, 1H, s; 6.90, 1H, t; 5.95, 3H,m; 3.18, 4H, rn; 1.95, 4H, m.

9.2 Synthesis of Pyrrolidinyl Ketone 216

A solution of aminophenol 214 (0.74 g, 4.6 mmol) and tetrafluorophthalicanhydride (1 g, 4.6 mmol) was refluxed in toluene (5 ml) for 3 hr. Thesolution was cooled to rt and the precipitated collected to yieldpyrrolidinyl ketone 216 (1.3 g, 77%).

9.3 Synthesis of Tetrafluoro 218

Phosphorous oxytrichloride (1 ml, 10 mmol) was added to a solution ofpyrrolidinyl ketone 216 (1.3 g, 3.5 mmol) in chloroform (10 ml). Thesolution was stirred for 15 min., aminophenol 214 (0.56 g. 3.5 mmol) wasadded and the solution was refluxed for 4 hr. The solution was cooled tort and the reaction quenched with water (0.5 ml). Tetrafluoro 218 waspurified by C18 reverse phase chromatography (MeOH/0.1 M TEAA, 4:1) toafford metallic green solid (0.73 g, 41%, Abs. max. 576 nm, Em. max 594nm, CH₃OH).

9.4 Synthesis of Rhodamine Dye 234

Rhodamine dye 234 (Abs. max 590 nm, Em. max 606 nm, CH₃OH; Em. max 61.1nm, H₂O) was synthesized from 218 as described in Section 7.2, supra,and purified by C-8 reverse phase HPLC, eluting with an acetonitrile/0.1M TEAA gradient.

10. Example Synthesis of Rhodamine Dye 236

Rhodamine dye 236 was synthesized from 3-(bisbenzylamino)phenol andtetrafluorophthalic anhydride 114 as illustrated in Schemes I and II.

10.1 Synthesis of Bis-Benzyl Tetrafluoro Ketone 222

A solution of 3-(bisbenzylamino)phenol (14.5 g, 50 mmol) andtetrafluorophthalic anhydride (11 g, 50 mmol) were refluxed in toluene(50 ml) for 18 hr. The solvent was evaporated and the residue purifiedby C18 reverse phase chromatography (MeOH/water, 7:3) to affordbis-benzyl tetrafluoro ketone 222 as a white solid (6.5 g, 25%).

10.2 Synthesis of Tetra-Benzyl Tetrafluoro 224

Phosphorous oxytrichloride (3.3 ml, 35 mmol) was added to a solution ofbis-benzyl ketone 222 (6.0 g, 12 mmol) in chloroform (60 ml). Thesolution was stirred for 15 min., 3-(bisbenzylamino)phenol (3.4 g, 12mmol) was added and the mixture was refluxed for 3 hr. The solution wascooled to room temperature and the reaction was quenched with water (1ml). The solvent was evaporated and the residue purified by C18 reversephase chromatography (MeOH/0.1 M TEAA, 9:1) to afford tetra-benzyltetrafluoro dye 224 as a dark red solid (0.62 g, 7%, Abs. max 566 nm).

10.3 Synthesis of Tetrafluoro 226

A suspension of dye 224 (0.62 g, 0.8 mmol) was heated in conc. HBr (20ml) to 110° C. for 45 min. The reaction mixture was poured intoice-water and the precipitated was collected and purified by C18 reversephase chromatography (CH₂OH:0.1M TEAA, 3:2) to afford debenzylated dye226 as a metallic green solid (100 mg, 31%, Abs. max 521 nm).

10.4 Synthesis of Rhodamine Dye 236

Rhodamine dye 236 was synthesized from 226 as described in Section 7.2,supra. Purification of 236 was accomplished on C18 silica gel withstepwise elution with 20-70% methanol vs. 0.1 M triethylammoniumacetate. The dye 236 eluted between 30% and 50% methanol. The solventwas evaporated and the residual blue oil was precipitated with ether toprovide the title dye as a blue solid (85 mg; Em. max 545 nm, H₂O).

11. Example Synthesis of FRET Dye 230

11.1 Synthesis of Succinimidyl (NHS) Ester of 196

To a solution of dye 196 (5 mg) in DMF (100 μl) was added succinimidyltetramethyluronium tetrafluoroborate (20 mg) and diisopropylamine (10μL). TLC analysis on C2-reverse phase silica gel eluting with 1:1methanol:0.1 M triethylammonium acetate could distinguish dye 196(Rf=0.2) from 196 succinimidyl ester (Rf=0). After 1 hr, the reactionappeared to be complete and was partitioned between 5% HCl anddichloromethane. The organic layer was dried over Na₂SO₄ and the solventevaporated to yield the 196 NHS ester as a blue solid.

11.2 Synthesis of FRET Dye 230

Energy-transfer (FRET) dye 230 is prepared by coupling 196 NHS ester and4-aminomethylbenzoic acid and 4′-aminomethyl-6-carboxyfluorescein(Molecular Probes Inc., Eugene, Oreg.), according to methods describedin U.S. Pat. No. 5,863,727. For example, 1 mmole of 196 NHS dissolved in250 μl of DMSO is added to a solution of 2 μmole4′-aminomethyl-6-carboxyfluorescein in 100 μl DMSO and 20 μltriethylamine. After mixing the solution is let stand for about 12hours, monitoring the progress of coupling by reverse phase HPLC.

12. Example Synthesis of FRET Dye 230-ddATP

FRET dye 230 is activated as the NHS ester by the method of Examplesupra 11.1 and coupled to 7-deaza-7-aminopropargyl, 2′-3′dideoxyadenosine-5′-triphosphate according to the methods in U.S. Pat.Nos. 5,821,356 and 5,770,716 to give FRET dye 230-ddATP. Alternatively,4′-trifluoroacetamidomethyl 6-carboxyfluorescein NHS ester is coupledwith 7-deaza-7-aminopropargyl, 2′-3′ dideoxyadenosine-5′-triphosphate togive the intermediate dye-nucleotide. The trifluoroacetyl protectinggroup is removed under basic conditions and the intermediate dyenucleotide is coupled with 230 NHS ester to give FRET dye 230-ddATP by atwo-step method of conjugating the energy-transfer dye to a nucleotide.

13. Example Sanger-Type Sequencing Using an Energy Transfer Dye

Following the methods described in U.S. Pat. Nos. 5,821,356 and5,366,860, Sanger-type terminator sequencing was performed on pGEM (PEBiosystems, Foster City, Calif.) using Taq FS polymerase (PE Biosystems,Foster City, Calif.), a mixture of four dNTPs, an unlabeled sequencingprimer and a single labeled terminator (6-FAM-230-ddATP). A plot of theresultant sequencing data, obtained on an ABI PRISM Model 310 instrument(PE Biosystems, Foster City, Calif.) is provided in FIG. 1.

14. Example Four-Color Sanger-Type Sequencing

Following the methods described in U.S. Pat. Nos. 5,821,356 and5,366,860, four-color Sanger-type terminator sequencing was performed onpGEM1 (PE Biosystems, Foster City, Calif.) using Taq FS polymerase (PEBiosystems, Foster City, Calif.), a mixture of four dNTPs, an unlabeledsequencing primer and a mixture of four, 3′-fluoro,spectrally-resolvable labeled terminators (6-FAM-230-ddATP;5-FAM-236-7-deaza-ddGTP; 6-FAM-JON-ddTTP; 6-FAM-ROX-ddCTP). A plot ofthe resultant sequencing data, obtained on an ABI PRISM Model 310instrument (PE Biosystems, Foster City, Calif.) is provided in FIG. 2.

As can be seen in FIG. 2, all of the dye-labeled polynucleotides exhibitsignificant fluorescence intensity. Moreover, the different dye-labeledpolynucleotide exhibit sufficiently similar mobilities, resulting ingood resolution.

15. Example Anti-Human Antibody-Dye Conjugate Detection

Anti-Human Il-8 Antibody-Dye-196 Conjugate

Polyclonal anti-human IL-8 antibody (R&D Systems, Minneapolis, Minn.;0.5 mg in 0.5 ml PBS) was incubated with 50 μl of 1 M Na₂CO₃ and 105 μgof dye 196 for 1 hour at room temperature, in the dark. Theantibody-dye-196 conjugate (Abs. max 666 nm, H₂O) was separated from thefree dye using a sephadex G-50 (fine) size exclusion column. Theconjugate was passed through a 0.2μ filter and stored at 4° C. in PBS.The concentration of the antibody was determined according to thefollowing equation: [A₂₈₀−(0.82×A₆₄₀)]/170,000, where 0.82=A₂₈₀/A₆₄₀ ofthe free dye and 170,000 is the extinction coefficient for the antibody.The concentration of the dye was determined according to the followingequation: A₆₄₀/120,000, where 120,000 is the extinction coefficient fordye 196. The dye 196-labeled polyclonal anti-human IL-8 antibodyconcentration was 0.18 mg/ml with an F/P ratio of 2.3/1.

Preparation of Monoclonal Anti-Human IL-8 Antibody Coated Beads

Goat anti-mouse IgG (Fc) polystyrene beads (200 ml of 0.5% w/v; 6 μmbead diameter; Spherotech) were first washed 3 times by centrifugationand resuspension of the bead pellet with 1 ml PBS. 4 μg of monoclonalanti-human IL-8 antibodies (R&D Systems) was added to the beadsuspension for a final volume of 1 ml. After incubation for 16 hrs atroom temperature with gentle mixing, the monoclonal antibody-coatedbeads were washed 2 times as described above and resuspended in 1 mlPBS. The final bead concentration was 0.1% w/v or 8×10⁶ beads/ml. Themonoclonal antibody-coated beads were stable for approximately 1 monthwhen stored at 4° C.

Fluorescent-Linked Immunosorbant Assay (FLISA)

To generate a standard curve, a two-fold serial dilution of human IL-8peptide (R&D Systems) in 50 μl FLISA buffer (PBS containing 1 mg/ml BSA,0.35 M NaCl, 0.1% Tween 20, and 0.01% NaN₃) were aliquoted into a96-well plate (Corning Costar No. 3904). Subsequently, 50 μl of amixture containing monoclonal anti-human IL-8 antibody-coated beads anddye 196-labeled polyclonal anti-human IL-8 antibodies in FLISA bufferwas then added to each well. The final concentrations in a total volumeof 100 μl per well were 64,000 antibody-coated beads/ml and 0.11 μg/mlof dye 196-labeled polyclonal anti-human IL-8 antibodies. The serialdilution for IL-8 peptide ranged from 2,000-2.0 pg/ml. The assaycontained eight replicates, and control wells containing no peptide wereincluded. After incubating overnight at room temperature in the dark,the 96-well plate was scanned using the FMAT 8100 HTS System (PEBiosystems, Foster City, Calif.), a macroconfocal imaging systemequipped with a 633-nm HeNe laser and 2-channel fluorescence detection(FL1: 650-685 nm, FL2: 685-720 nm). A 1 mm² area of each well is rasterscanned at a depth of focus of 100 μm and at a rate of 1 sec/well, with256 scan lines generated. The fluorescence intensity associated witheach bead is then obtained from the digitized images and the averagefluorescence intensity per bead is calculated per well. A log vs loggraph of the average fluorescence intensity in FL1 vs pg/ml of the IL-8peptide is shown in FIG. 4. The linear dynamic range of the assay is250-3.9 pg/ml IL-8.

While the present invention has been described by reference to thepreferred embodiments and examples detailed above, it is to beunderstood that these examples are intended in an illustrative, ratherthan limiting, sense. It is contemplated that modifications that do notdepart from the spirit of the invention will readily occur to those ofskill in the art. Any such modifications are intended to fall within thescope of the appended claims.

All of the references, publications and patents cited in thespecification are incorporated herein by reference to the same extent asif each reference were individually incorporated by reference.

1. A rhodamine dye or a salt thereof, comprising a rhodamine-type parentxanthene ring having attached to the xanthene C9 carbon a phenyl groupthat is further substituted with an ortho carboxy or ortho sulfonategroup or a salt thereof, one to three substituted or unsubstitutedaminopyridinium groups and a substituted or unsubstituted alkylthio,arylthio or heteroarylthio group, wherein the rhodamine dye comprisesthe structure:

wherein: n is an integer selected from the group consisting of 1, 2, and3; Y is a rhodamine-type parent xanthene ring attached to theillustrated phenyl group at the xanthene C9 carbon; each R isindependently selected from the group consisting of a (C₁-C₆) alkyl orheteroalkyl, a (C₅-C₂₀) aryl or heteroaryl, a (C₆-C₂₆) arylalkyl orheteroalkyl and a (C₅-C₂₀) arylalkyl or heteroaryl-heteroalkyl, or whentaken together, both R form a moiety selected from the group consistingof a (C₄-C₁₀) alkyldiyl, a (C₄-C₁₀) alkyleno, a heteroalkyldiyl and aheteroalkyleno; S is sulfur; Z is selected from the group consisting ofa (C₁-C₁₂) alkyl, a (C₁-C₁₂) alkyl substituted with one or more of thesame or different W¹ groups, a (C₅-C₂₀) aryl or heteroaryl and a(C₅-C₂₀) aryl or heteroaryl substituted with one or more of the same ordifferent W² groups; W¹ is selected from the group consisting of —X, —R,═O, —OR, —SR, ═S, —NRR, ═NR, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO,—NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R,—C(S)X, —C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRRand —C(NR)NRR; W² is selected from the group consisting of —R, —OR, —SR,—NRR, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X,—C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and—C(NR)NRR; each X is independently a halogen; and Y or Z is optionallysubstituted with L, wherein L is a bond or a linker selected from thegroup consisting of a hydrophobic moiety, a charged group, a member of apair of specific binding molecules, a photo-activatable group and areactive functional group.
 2. The rhodamine dye of claim 1, wherein Zhas the form Z¹-L-R_(x), or a salt thereof, wherein: Z¹ is selected fromthe group consisting of a (C₁-C₁₂) alkyldiyl, a (C₁-C₁₂) alkyldiylindependently substituted with one or more of the same or different W¹groups, a (C₅-C₁₄) aryldiyl or heterodiyl and a (C₅-C₁₄) aryldiyl orheterodiyl independently substituted with one or more of the same ordifferent W² groups; L is selected from the group consisting of a bondand a linker; and R_(x) is a reactive functional group.
 3. The rhodaminedye of claim 2, in which Y is selected from the group consisting of:

and a salt thereof, wherein: each of R¹ and R² when taken alone, isindependently selected from the group consisting of hydrogen and a(C₁-C₆) alkyl; each of R³ and R^(3′) when taken alone, is independentlyselected from the group consisting of hydrogen, a (C₁-C₆) alkyl, a(C₅-C₁₄) aryl and an arylaryl, or when taken together are a (C₄-C₆)alkyldiyl or an alkyleno, or when individually taken together with R² orR⁴ is a (C₂-C₆) alkyldiyl and a (C₂-C₆) alkyleno; R⁴, when taken alone,is selected from the group consisting of hydrogen and a (C₁-C₆) alkyl,or when taken together with R³ or R^(3′) is selected from the groupconsisting of a (C₂-C₆) alkyldiyl and an alkyleno; R⁵, when taken alone,is selected from the group consisting of hydrogen and a (C₁-C₆) alkyl,or when taken together with R⁶ or R^(6′) is selected from the groupconsisting of a (C₂-C₆) alkyldiyl and an alkyleno; each of R⁶ and R^(6′)when taken alone, is independently selected from the group consisting ofhydrogen, a (C₁-C₆) alkyl, a (C₅-C₁₄) aryl and an arylaryl, or whentaken together are a (C₄-C₆) alkyldiyl or an alkyleno, or whenindividually taken together with R⁵ or R⁷ is a (C₂-C₆) alkyldiyl or analkyleno; R⁷, when taken alone, is selected from the group consisting ofhydrogen and a (C₁-C₆) alkyl, or when taken together with R⁶ or R^(6′)is a (C₂-C₆) alkyldiyl or an alkyleno; R⁸, when taken alone, is selectedfrom the group consisting of hydrogen and a (C₁-C₆) alkyl; R⁹ indicatesthe point of attachment to the ortho-carboxyphenyl bottom ring; and eachof R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ isindependently selected from the group consisting of hydrogen and a(C₁-C₆) alkyl, or R¹⁰, R¹¹, R¹² and R¹³ taken together are a (C₅-C₁₄)aryleno or a (C₅-C₁₄) aryleno substituted with one or more of the sameor different (C₁-C₆) alkyl(s), or R¹⁸, R¹⁹, R²⁰ and R²¹ taken togetherare a (C₅-C₁₄) aryleno or an aryleno substituted with one or more of thesame or different (C₁-C₆) alkyl(s).
 4. The rhodamine dye of claim 3,wherein R², when taken together with R³ or R^(3′) is a (C₂-C₆) alkyldiylor an alkyleno.
 5. The rhodamine dye of claim 4, wherein: alkyl isselected from the group consisting of methyl, ethyl and a propyl; arylis selected from the group consisting of phenyl and naphthyl; arylarylis biphenyl; alkyldiyl or alkyleno bridges formed by taking R² togetherwith R³ or R^(3′), R⁷ together with R⁶ or R^(6′), or R⁴ together with R³or R^(3′), are selected from the group consisting of ethano, a propano,1,1-dimethylethano, 1,1-dimethylpropano and 1,1,3-trimethylpropano;aryleno bridges formed by taking R¹ together with R² are selected fromthe group consisting of benzo and naphtho; alkyldiyl or alkyleno bridgeformed by taking R³ together with R^(3′), or R⁶ together with R^(6′), isa butano; alkyldiyl or alkyleno bridges formed by taking R⁵ togetherwith R⁶ or R^(6′) are selected from the group consisting of ethano, apropano, 1,1-dimethylethano, 1,1-dimethylpropano and1,1,3-trimethylpropano; and aryleno bridge formed by taking R¹⁰, R¹¹,R¹² and R¹³ together, or R¹⁸, R¹⁹, R²⁰ and R²¹ together, is benzo. 6.The rhodamine dye of claim 4, wherein L is a bond.
 7. The rhodamine dyeof claim 2, wherein R_(x) is selected from the group consisting ofcarboxyl, carboxylate, an ester and an activated ester.
 8. The rhodaminedye of claim 2, wherein Z¹ is selected from the group consisting of a(C₁-C₁₂) alkyleno, a (C₁-C₁₂) alkano, a (C₅-C₁₀) aryldiyl orheteroaryldiyl, phenyldiyl, phena-1,4-diyl, naphthadiyl,naphtha-2,6-diyl, pyridindiyl and purindiyl.
 9. The rhodamine dye ofclaim 2, wherein Y is selected from the group consisting of:


10. The rhodamine dye of claim 2, wherein L is a bond.
 11. The rhodaminedye of claim 2 which comprises the structure:

or a salt thereof.
 12. The rhodamine dye of claim 11, wherein Y isselected from the group consisting of Y-1, Y-2, Y-3 and Y-4.
 13. Therhodamine dye of claim 11, wherein Y is selected from the groupconsisting of Y-20a, Y-21a, Y-22a, Y-23a, Y-24a, Y-25a, Y-31a, Y-34a,Y-35a, Y-36a, Y-39a, Y-41a, Y-42a, Y-43a, Y-44a, Y-45a and Y-46a. 14.The rhodamine dye of claim 1 having the structure:

wherein: Y¹ is a rhodamine-type parent xanthene ring attached to theillustrated phenyl group at the xanthene C9 carbon; L is a bond orlinker attached to a xanthene nitrogen atom or a xanthene C4 carbon; andR_(x) is a reactive functional group.
 15. The rhodamine dye of claim 14,wherein Y¹ is selected from the group consisting of:

wherein the dashed line at the nitrogen or C4 atom indicates the pointof attachment of L.
 16. The rhodamine dye of claim 15, wherein: alkyl isselected from the group consisting of methanyl, ethanyl and propanyl;aryl is selected from the group consisting of phenyl and naphthyl;arylaryl is biphenyl; alkyldiyl or alkyleno bridges formed by taking R²together with R³ or R^(3′), R⁷ together with R⁶ or R^(6′), or R⁴together with R³ or R^(3′), are selected from the group consisting ofethano, a propano, 1,1-dimethylethano, 1,1-dimethylpropano and1,1,3-trimethylpropano; aryleno bridges formed by taking R¹⁰, R¹¹, R¹²and R¹³ together or R¹⁸, R¹⁹, R²⁰ and R²¹ together are benzo.
 17. Therhodamine dye of claim 14, wherein L is selected from the groupconsisting of a (C₁-C₆) alkyldiyl, a (C₁-C₆) alkano, a (C₅-C₂₀)aryldiyl, phenyldiyl, phena-1,4-diyl, naphthyldiyl, naphtha-2,6-diyl,naphtha-2,7-diyl, a (C₆-C₂₆) arylalkyldiyl, —(CH₂)_(i)-φ- and—(CH₂)_(i)-Ψ-, where each i is independently an integer having the valuebetween 1 and 6, φ is selected from the group consisting of a (C₅-C₂₀)aryldiyl, phenyldiyl and phena-1,4-diyl and Ψ is selected from the groupconsisting of naphthyldiyl, naphtha-2,6-diyl and naphtha-2,7-diyl. 18.The rhodamine dye of claim 14, wherein R_(x) is selected from the groupconsisting of carboxyl, carboxylate, an ester and an activated ester.19. The rhodamine dye of claim 14, wherein Z is selected from the groupconsisting of a (C₁-C₁₂) alkyl, a (C₁-C₁₂) alkanyl, a (C₅-C₁₀) aryl orheteroaryl, phenyl, naphthyl, naphth-1-yl, naphth-2-yl, pyridyl andpurinyl.
 20. The rhodamine dye of claim 14, wherein Y¹ is selected fromthe group consisting of:

wherein the dash at the nitrogen or C4 atom indicates the point ofattachment of L.
 21. The rhodamine dye of claim 14 having the structure:

or a salt thereof.
 22. The rhodamine dye of claim 21, wherein Y¹ isselected from the group consisting of Y-1b, Y-2b, Y-3b, Y-4-b, Y-1c,Y-2c, Y-3c and Y-4-c.
 23. The rhodamine dye of claim 21, wherein Y¹ isselected from the group consisting of Y-20b, Y-20c, Y-21b, Y-21c, Y-22b,Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c, Y-34b,Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b, Y-39c, Y-41c, Y-42b,Y-43b, Y-43c, Y-46b and Y-46c.
 24. An energy-transfer dye paircomprising a donor dye linked to an acceptor dye, wherein the donor dyeor the acceptor dye is a compound according to claim 1 and either orboth of said donor and acceptor dyes include an optional linking moiety.25. The dye pair of claim 24 having the structure:

or a salt thereof, wherein: R⁴¹ is a covalent linkage formed uponreaction between a nucleophile and an electrophile; L″ is selected fromthe group consisting of a bond and a linker; n is an integer selectedfrom the group consisting of 1, 2, and 3; and DD/AD is a donor dye or anacceptor dye which includes a linking moiety.
 26. The dye pair of claim25, wherein Y is selected from the group consisting of Y-1, Y-2, Y-3,Y-4, Y-20a, Y-21a, Y-22a, Y-23a, Y-24a, Y-25a, Y-31a, Y-34a, Y-35a,Y-36a, Y-39a, Y-41a, Y-42a, Y-43a, Y-44a, Y-45a and Y-46a.
 27. The dyepair of claim 25, wherein L is a bond.
 28. The dye pair of claim 25,wherein R⁴¹ has the formula —C(O)NR⁴⁵—, wherein R⁴⁵ is selected from thegroup consisting of hydrogen and a (C₁-C₆) alkyl.
 29. The dye pair ofclaim 25, wherein Z¹ is selected from the group consisting of a (C₁-C₁₂)alkyleno, a (C₁-C₁₂) alkano, a (C₅-C₁₀) aryldiyl or heteroaryldiyl,phenyldiyl, phena-1,4-diyl, naphthadiyl, naphtha-2,6-diyl, pyridindiyland purindiyl.
 30. The dye pair of claim 25, wherein L″ is—R⁴³—Z³—C(O)—R⁴⁴—R⁴⁵—, wherein R⁴³ is a (C₁-C₆) alkyldiyl, and is bondedto R⁴², wherein R⁴² is selected from the group consisting of O, S andNH; Z³ is selected from the group consisting of a 5-6 membered cyclicalkenyldiyl or heteroalkenyldiyl and a (C₅-C₁₄) aryldiyl orheteroaryldiyl; R⁴⁴ is selected from the group consisting of O, S andNH; and R⁴⁵ is a (C₁-C₆) alkyldiyl.
 31. The dye pair of claim 25,wherein DD/AD is a fluorescein dye in which the linking moiety is areactive functional group, and wherein L″ is attached to the fluoresceindye at the xanthene C4 carbon.
 32. The dye pair of claim 25 having thestructure:

wherein R⁵⁰ is carboxyl, a salt, an ester or an activated ester thereof.33. The dye pair of claim 32, wherein Y is selected from the groupconsisting of Y-1, Y-2, Y-3, Y-4, Y-20a, Y-21a, Y-22a, Y-23a, Y-24a,Y-25a, Y-31a, Y-34a, Y-35a, Y-36a, Y-39a, Y-41a, Y-42a, Y-43a, Y-44a,Y-45a and Y-46a.
 34. The dye pair of claim 24 having the structure:

wherein: R⁴¹ is a covalent linkage formed upon reaction between anucleophile and an electrophile; L″ is selected from the groupconsisting of a bond and a linker; n is an integer selected from thegroup consisting of 1, 2, and 3; and DD/AD is a donor dye or an acceptordye which includes a linking moiety.
 35. The dye pair of claim 34,wherein Y¹ is selected from the group consisting of Y-1b, Y-2b, Y-3b,Y-4-b, Y-1c, Y-2c, Y-3c, Y-4-c, Y-20b, Y-20c, Y-21b, Y-21c, Y-22b,Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c, Y-34b,Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b, Y-39c, Y-41c, Y-42b,Y-43b, Y-43c, Y-46b and Y-46c.
 36. The dye pair of claim 34, wherein Lis selected from the group consisting of a (C₁-C₆) alkyldiyl and a(C₁-C₃) alkano.
 37. The dye pair of claim 34, wherein R⁴¹ is an amide ofthe formula —C(O)NR⁴⁵—, wherein R⁴⁵ is selected from the groupconsisting of hydrogen and a (C₁-C₆) alkyl.
 38. The dye pair of claim34, wherein Z is selected from the group consisting of a (C₁-C₁₂) alkyl,a (C₁-C₁₂) alkanyl, a (C₅-C₁₀) aryl or heteroaryl, phenyl, naphthyl,naphth-1yl, naphth-2-yl, pyridyl and purinyl.
 39. The dye pair of claim34, wherein L″ is —R⁴³—Z³—C(O)—R⁴⁴—R⁴⁵—, wherein R⁴³ is a (C₁-C₆)alkyldiyl and is bonded to R⁴², wherein R⁴² is selected from the groupconsisting of O, S and NH; Z³ is selected from the group consisting of a5-6 membered cyclic alkenyldiyl or heteroalkenyldiyl and a (C₅-C₁₄)aryldiyl or heteroaryldiyl; R⁴⁴ is selected from the group consisting ofO, S and NH; and R⁴⁵ is a (C₁-C₆) alkyldiyl.
 40. The dye pair of claim34, wherein DD/AD is a fluorescein dye in which the linking moiety is areactive group R_(x) and wherein L″ is attached to the fluorescein dyeat the xanthene C5 carbon.
 41. The dye pair of claim 34 having thestructure:

wherein: Y¹ is selected from the group consisting of Y-20b, Y-20c,Y-21b, Y-21c, Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c,Y-31b, Y-31c, Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b,Y-39c, Y-41c, Y-42b, Y-43b, Y-43c, Y-46b and Y-46c and R⁵⁰ is carboxyl,a salt, an ester or an activated ester thereof.
 42. The dye pair ofclaim 35, wherein Y¹ is selected from the group consisting of Y-1b,Y-2b, Y-3b, Y-4-b, Y-1c, Y-2c, Y-3c, Y-4-c, Y-20b, Y-20c, Y-21b, Y-21c,Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c,Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b, Y-39c, Y-41c,Y-42b, Y-43b, Y-43c, Y-46b and Y-46c.
 43. A polynucleotide labeled witha rhodamine dye according to claim 2 or an energy-transfer dye pairaccording to claim
 28. 44. A method of generating a labeled primerextension product, comprising the step of enzymatically extending aprimer-target hybrid in the presence of a mixture ofenzymatically-extendable nucleotides capable of supporting continuousprimer extension and a terminator, wherein said primer or saidterminator is labeled with a rhodamine dye according to claim 2 or anenergy-transfer dye pair according to claim
 28. 45. The method of claim44, wherein the terminator has the structure:

wherein each of R⁷⁰ and R⁷¹, when taken alone, is independently selectedfrom the group consisting of hydrogen, a halide and any moiety whichblocks polymerase-mediated template-directed polymerization.
 46. Themethod of claim 44, wherein the terminator is a mixture of fourdifferent terminators, one which terminates at a template A, one whichterminates at a template G, one which terminates at a template C and onewhich terminates at a template T or U.
 47. The method of claim 44,wherein each of the four different terminators is labeled with adifferent, spectrally-resolvable fluorophore.